Nitrogen uptake in plants

ABSTRACT

This disclosure provides transgenic plants, including crop plants and methods for their production. The transgenic plants comprise recombinant DNA constructs for the expression of polypeptides encoded by the DNA constructs. The polypeptides are capable of conferring improved traits to the transgenic plants when expressed under the control of a tissue-enhanced promoter. This disclosure also pertains to transgenic plants and their progeny plants, wherein the transgenic or progeny plants comprise the recombinant DNA constructs and are selected for having enhanced nitrogen use efficiency and/or nitrogen uptake. Seed of the transgenic plants that can be grown into a plant that comprise the disclosed recombinant DNA constructs and exhibits having enhanced nitrogen use efficiency, and which may be selected for this trait, are also envisioned.

FIELD OF THE INVENTION

The present description relates to compositions and methods forimproving nitrogen use efficiency in plants, for example, by improvingnitrogen uptake or assimilation efficiency.

BACKGROUND

Nitrogen is a critical limiting nutrient for plants. Nitrogen fertilizeris a significant contributor to the yield increases obtained in the lastseveral decades. However, these yield benefits have monetary andenvironmental costs, and nitrogen-based fertilizer represents asignificant fraction of a farmer's input costs. Furthermore, crops onlyuse a fraction of applied nitrogen. For example, it has been estimatedthat 50-70% of the nitrogen provided to the soil is lost(Masclaux-Daubresse et al., 2010, Ann. Bot. 105: 1141-1157; Hodge etal., 2000, Trends Plant Sci. 5: 304-308). Maize production in the US isreported to have a nitrogen fertilizer recovery efficiency of 37%(Cassman et al., 2002, Ambio 31: 132-140), and increased fertilizerapplication rates are subject to diminishing returns. A hectare of corn,for example, retains 39% of the first 100 kilograms of nitrogen appliedas fertilizer, but only 13% of the second 100 kilograms of nitrogenapplied (Socolow, 1999, Proc. Natl. Acad. Sci. USA 96: 6001-6008). As aconsequence, nitrogen fertilizer that is not taken up by plants isgenerally lost as runoff or converted to nitrogen gases by microbialaction, contributing to water and air pollution.

Thus, improving their efficiency of a crop plant's nitrogen use (i.e.,its Nitrogen Use Efficiency, or N Use Efficiency, or NUE) would have thebenefit of improving yield and agricultural sustainability whilereducing negative environmental impact. NUE has been defined asincreased grain yield per unit nitrogen available from the soil(Masclaux-Daubresse et al., 2010, supra), and thus it is judicious toidentify means to increase the grain yield that may be obtained per unitnitrogen available from the soil.

Plants obtain nitrogen through the processes of uptake and assimilation(Buchanan et al., 2000, Biochemistry and Molecular Biology of Plants,American Society of Plant Physiologists, Rockville, Md.;Masclaux-Daubresse et al., 2010, supra). Uptake refers to the transportof nitrogen into the plant, and assimilation is the conversion ofnitrate and ammonia to amino acids. Plants generally take up nitrogenfrom the soil in the form of nitrate or ammonium. Plants contain bothlow affinity and high affinity transport systems for these ions. In thecase of nitrate, there is both a constitutive and an inducible highaffinity transport system (Glass et al., 2002, J. Exp. Bot. 53:855-864). Once nitrate crosses the plasma membrane, it is eithermetabolized in the cytoplasm of root cells or transported to the shootvia the xylem. For example, in wheat, up to 80% of the absorbed nitrateis reduced within the leaves (Ashley et al., 1975, Plant Physiol. 55:1102-1106). Nitrate is reduced to ammonia through the action of nitratereductase and nitrite reductase. Assimilation of ammonia takes placethrough the glutamine synthetase/glutamine-2-oxoglutarateaminotransferase (GS/GOGAT) pathway. Glutamine synthetase (GS) adds anamino group to glutamate to make glutamine, and GOGAT transfers theamino group to α-ketoglutarate to make a second molecule of glutamate.Photosynthesis provides the fixed carbon, energy, and reductantnecessary for assimilation.

Plant nitrogen use efficiency could conceivably be increased by severalmechanisms (Lawlor 2002, J. Exp. Bot. 53: 773-787). One mechanism couldbe increasing nitrogen uptake (which can be defined as the percentage ofapplied nitrogen taken up by plants (Maust and Williamson, 1994, J.Amer. Soc. Hort. Sci., 119: 195-201), through higher root surface area,deeper penetration into the soil, or more high affinity nitrate orammonium transporters. A second mechanism could be increasedassimilation, possibly by increased activity of assimilatory enzymes orremoval of negative regulation. Nitrogen utilization or assimilationefficiency, NUtE, is the fraction of plant-acquired nitrogen to beconverted to total plant biomass or grain yield; (Xu et al., 2012, Annu.Rev. Plant Biol. 63:153-182). A third mechanism could be increasedcapacity to store nitrogen when it is available. Nitrogen is stored inthe form of nitrate in cell vacuoles, but stored nitrate supplies areexhausted in a matter of days (Glass et al., 2002, supra). Nitrogen isalso stored in the form of amino acids and protein, and this storage isdependent upon sufficient carbon availability. Control of nitrogenlosses is also possible. Nitrate and ammonia exit as well as enter rootcells. Photorespiration is another source of ammonia loss Ammoniareleased through photorespiration is recycled through the GS/GOGATpathway, but this process may not be fully efficient. Overexpression ofcytosolic glutamine synthetase in tobacco increased biomass produced,presumably through increased efficiency of ammonia recycling (Oliveiraet al., 2002, Plant Physiol. 129: 1170-1180). The intrinsic nitrogen useefficiency (defined as biomass produced per unit N) could be changed bychanging the plant's fundamental carbon/nitrogen ratio. Improving theNUE of crop plants has the potential to reduce fertilizer applicationrates, providing both cost savings and environmental benefits.

In spite of the apparent advantages of improved NUE, decades of researchhave not produced significant improvements in NUE in crops, and improvedNUE is largely an unmet need in agriculture today.

The present description relates to methods and compositions forproducing transgenic plants with modified traits, particularly traitsthat address agricultural and food needs by improving nitrogen useefficiency. In addition to reducing the demand for nitrogen application,it is expected that improving nitrogen use efficiency will improve yieldand may provide significant value by allowing the plant to thrive inhostile environments, where, for example, low nutrient availability maylimit yield or diminish or prevent growth of non-transgenic plants.

In this description, the expression levels of certain polynucleotide andpolypeptide sequences identified herein may be manipulated to produceimproved yield in commercially valuable plants and crops as well. Otheraspects and embodiments of the description are described below and canbe derived from the teachings of this disclosure as a whole.

SUMMARY OF THE DISCLOSURE

The present disclosure is directed to a method for producing a plantthat has improved or enhanced nitrogen uptake and/or assimilation in theplant or in a part of the plant (for example, in roots or leaves;)relative to a control plant or its corresponding or analogous part. Inthis method, a plant is grown in a medium that contains either alimiting concentration of nitrogen that limits growth of the plant (forexample, 2 mM total nitrogen in the medium) or an ample concentration ofnitrogen that does not limit growth of the plant (for example, 10 mMtotal nitrogen in the medium). Expression analysis of the plant may thenreveal the presence of one or more polypeptides (the “instantpolypeptides”) that have a higher level of expression when the plant isgrown in the limiting nitrogen medium as compared to plants grown in themedium with ample nitrogen. Genes may also be identified that, whenoverexpressed, produce changes in growth (e.g. enhanced root growth) orappearance (e.g. darker green leaves) in plate based assays using mediawith altered nitrogen or carbon content, or increased growth, biomass,nitrogen content, or photosynthetic capacity in soil-grown plants, andtested for the ability to confer enhanced nitrogen uptake and/orassimilation. The expression of a polypeptide identified in this mannermay be regulated by a developmentally-regulated promoter. For example,expression of the promoter may be enhanced in root, root cap, rootmeristem, root vasculature, vascular, and/or green tissue; that is, theactivity of the promoter is enhanced in one or more of these tissuesrelative to other plant tissues). It is anticipated that transformedplants that comprise one or more nucleic acid constructs that containthe tissue-enhanced promoter and a polynucleotide that encodes one ofthe instant polynucleotide will have greater nitrogen uptake and/orassimilation, as measured by such parameters as Nitrogen UptakeEfficiency (NUpE) or Usage Index (UI) relative to a control plant and asa result of the expression of the polynucleotide. The one or morenucleic acid constructs may be introduced into the plant by, forexample, transformation or breeding. In this method, a regulator of geneexpression may be identified that can enhance nitrogen uptake whenexpression of the regulator is enhanced in root, root cap, rootmeristem, root vasculature, vascular, and/or green tissue of the plantor a part of the plant. In this method, a transformed plant may beselected that has greater nitrogen uptake than the control plant.

The converse observation, in which expression analysis of the plantidentifies one or more endogenous polypeptides that have a higher levelof expression when the plant is grown in the ample nitrogen medium ascompared to plants grown in the medium with limiting nitrogen, may beused to identify one or more endogenous polypeptides that may enhancenitrogen uptake and/or assimilation when expression of the endogenouspolypeptide(s) is/are down-regulated. Down-regulation of expression maybe accomplished with a means that suppresses transcription ortranslation of the endogenous polypeptide. Of some interest aresuppressors of gene expression such as, for example, an RNAi molecule,an siRNA molecule, an antisense molecule, a ribozyme molecule, adeoxyribosyme molecule (a “DNAzyme”) or a triple helix molecule thatdecreases the expression of the endogenous polypeptide. Gene expressionsuppressors may be introduced into a plant by breeding plants with aparental line that contains an instant gene expression suppressor, or bydirect application or, in a desirable embodiment, by way of a nucleicacid construct that encodes the suppressor. It is anticipated thatplants that comprise nucleic acid constructs encoding one or more of theinstant suppressors will suppress or inhibit the activity of an instantpolypeptide in the plant and thereby enhance nitrogen uptake in theplant. In this manner, a regulator of gene expression that can suppressprotein expression or protein activity of the plant or a part of theplant may be identified.

The instant disclosure is also directed to a method for enhancingnitrogen uptake in a crop plant relative to a control plant by providinga transformed crop plant that comprises at least one of the instantrecombinant nucleic acid constructs, and the construct or constructscomprise a tissue enhanced promoter that preferentially drivesexpression in root, root cap, root meristem, root vasculature, vascular,and/or green tissue, and in the same construct or a separate construct,an operably-linked polynucleotide the expression of which is regulatedby the promoter. In this context, “providing” may refer to, for example,any one of the art-recognized means to introduce a nucleic acidconstruct into a plant or plant cell, such as by transformation orbreeding where at least one parent line comprises at least one of theinstant nucleic acid constructs (two parental lines may each contain aninstant nucleic acid construct, as in the case when one plant linecomprises a tissue-enhanced promoter that regulates expression of apolynucleotide comprised within a second promoter comprised within adifferent parental plant line). The polynucleotide encodes a polypeptidethat is at least 30%, at least 31%, at least 32%, at least 33%, at least34%, at least 35%, at least 36%, at least 37%, at least 38%, at least39%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 90%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%,or about 100% identical to SEQ ID NO:2n, where n=1 to 1131, oralternatively expressed as any of SEQ ID NOs: 2, 4, 6, 8, or any eveninteger to 2262. The tissue-enhanced promoter preferentially enhancesexpression of the polynucleotide in root, root cap, root meristem, rootvasculature, vascular, and/or green tissue in the transformed plant orin a part of the transformed plant, and the preferential enhancement ofexpression results in increased nitrogen uptake in the transformed plantrelative to the control plant.

Another aspect of the instant disclosure is a method of producing a cropplant with enhanced nitrogen uptake by providing a crop plant that has astably-integrated, recombinant DNA construct comprising a promoter thatis functional in plant cells and operably linked to DNA that encodes orsuppresses a polypeptide presented in the Sequence Listing, or any ofSEQ ID NOs: 2n, where n=1 to 1131, wherein the expression and activityof the polypeptide confers enhanced nitrogen uptake relative to acontrol plant. The methods further comprise producing seed and a progenyplant from the crop plant with enhanced nitrogen uptake, wherein theseed or progeny plant comprise the stably-integrated, recombinant DNAconstruct and the progeny plant or a plant grown from the seed exhibitenhanced nitrogen uptake relative to a control plant.

The instant disclosure also pertains to a recombinant nucleic acidconstruct comprising a root, root cap, root meristem, root vasculature,vascular, and/or green tissue-enhanced promoter that regulatesexpression of a polynucleotide, wherein the polynucleotide encodes apolypeptide is at least 30%, at least 31%, at least 32%, at least 33%,at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, atleast 39%, at least 40%, at least 41%, at least 42%, at least 43%, atleast 44%, at least 45%, at least 46%, at least 47%, at least 48%, atleast 49%, at least 50%, at least 51%, at least 52%, at least 53%, atleast 54%, at least 55%, at least 56%, at least 57%, at least 58%, atleast 59%, at least 60%, at least 61%, at least 62%, at least 63%, atleast 64%, at least 65%, at least 66%, at least 67%, at least 68%, atleast 69%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 90%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% or 96%, at least 97%, at least 98%, or at least99%, or about 100% identical to any of SEQ ID NOs: 2, 4, 6, 8, or anyeven integer to 2262.

The instant disclosure also pertains to a transformed crop plantproduced by any of the above described, methods, wherein the crop planthas enhanced nitrogen uptake relative to a control plant when theexpression of an introduced or endogenous polypeptide provided in thesequence listing is enhanced or inhibited, respectively.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

The Sequence Listing provides exemplary polynucleotide and polypeptidesequences of the instant description. The traits associated with the useof the sequences are included in the Examples.

Incorporation of the Sequence Listing.

The Sequence Listing provides exemplary polynucleotide and polypeptidesequences. The copy of the Sequence Listing, being submittedelectronically with this patent application, provided under 37 CFR §1.821-1.825, is a read-only memory computer-readable file in ASCII textformat. The Sequence Listing is named “MPS-0231P_ST25.txt,” theelectronic file of the Sequence Listing was created on Mar. 5, 2014, andis 8,181,555 bytes in size (7.80 megabytes in size as measured inMS-WINDOWS). The Sequence Listing is herein incorporated by reference inits entirety.

DETAILED DESCRIPTION

The present description relates to polynucleotides and polypeptides formodifying phenotypes of plants, particularly those associated withincreased photosynthetic resource use efficiency and increased yieldwith respect to a control plant (for example, a wild-type plant).Throughout this disclosure, various information sources are referred toand/or are specifically incorporated. The information sources includescientific journal articles, patent documents, textbooks, and internetentries. While the reference to these information sources clearlyindicates that they can be used by one of skill in the art, each andevery one of the information sources cited herein are specificallyincorporated in their entirety, whether or not a specific mention of“incorporation by reference” is noted. The contents and teachings ofeach and every one of the information sources can be relied on and usedto make and use embodiments of the instant description.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include the plural reference unless the context clearlydictates otherwise. Thus, for example, a reference to “a host cell”includes a plurality of such host cells, and a reference to “a plant” isa reference to one or more plants, and so forth.

Definitions

“Upregulation” or “up-regulation” refers to a process in which a cell oran organism (e.g., a plant) increases the quantity of a cellularcomponent, such as RNA or protein, in response to an internal orexternal signal. Upregulation may result in a greater activity ofinterest occurring in the cell or organism, for example, an increase innitrogen uptake. Conversely, “downregulation” or “down-regulation”refers to a process by which a cell decreases the quantity of a cellularcomponent, such as RNA or protein, in response to an internal orexternal signal. An internal or external signal may refer to, forexample, an environmental variable such as a particular stress or adevelopmental marker such as molecule that signals the onset oroccurrence of germination, root establishment, seedling growth, leafproduction and canopy development, leaf senescence, reproduction(fertilization and seed development) or an environmental stress such aslow environmental nitrogen, phosphorus, sulfur, iron, or potassium, saltstress, water stress, heat stress.

Tissue-specific, tissue-enhanced (that is, tissue-preferred), celltype-specific, and inducible promoters constitute non-constitutivepromoters. Promoters under developmental control include promoters thatpreferentially initiate transcription in certain tissues, such as xylem,leaves, roots, or seeds. Such promoters are examples of tissue-enhancedor tissue-preferred promoters (see U.S. Pat. No. 7,365,186).Tissue-enhanced promoters can be found upstream and operatively linkedto DNA sequences normally transcribed in higher levels in certain planttissues or specifically in certain plant tissues, respectively.“Cell-enhanced”, “tissue-enhanced”, or “tissue-specific” regulation thusrefer to the control of gene or protein expression, for example, by apromoter, which drives expression that is not necessarily totallyrestricted to a single type of cell or tissue, but where expression iselevated in particular cells or tissues to a greater extent than inother cells or tissues within the organism, and in the case oftissue-specific regulation, in a manner that is primarily elevated in aspecific tissue. Tissue-enhanced or preferred promoters have beendescribed in, for example, U.S. Pat. No. 7,365,186, U.S. Pat. No.7,619,133, and by Noh and Amasino, 1999. Plant Molec. Biol. 41:181-194.Generally, root-specific, root cap-specific, root meristem-specific,root vasculature-specific, vascular-specific, and/or greentissue-specific promoters are transcriptionally active entirely oralmost entirely in root, root cap, root meristem, root vasculature,vascular, and green tissue, respectively. Root-preferred or -enhanced,root cap-preferred or -enhanced, root meristem-preferred or -enhanced,root vasculature-preferred or -enhanced, vascular-preferred or-enhanced, and green tissue-preferred or -enhanced promoters aretranscriptionally active predominantly in one or more of these tissues,but are not necessarily expressed only in these tissue. A root-specific,enhanced or preferred promoter may be preferentially active during rootdevelopment and/or during germination. Examples of tissue-enhancedpromoters are found in the present Sequence Listing, in Table 3, or havebeen taught in, for example, US patent publication U520130305414 or byQing Qu and Takaiwa, 2004. Plant Biotechnol. J. 2:113-125).

An “inducible promoter” initiates transcription in response to anenvironmental stimulus such as a an external physical stimulus, forexample, abiotic stimuli including energy or a particular chemical orclass of chemicals, or a biotic stimulus, for example, a pathogen, or aninternal stimulus such as one or more markers that signal a stage ofdevelopment. Examples include “pathogen-inducible” promoters thatinitiate transcription in response to the presence of various pathogenicorganisms or their products, and developmentally-induced promoters thatare activated when a plant or plant part is at a particular growthstage, for example, “senescence-enhanced” (also referred to as“senescence-inducible”) promoters. Senescence-enhanced promoters areactive late in the life cycle of a plant during or near the time ofsenescence (Noh and Amasino, 1999. supra), and preferentially regulateexpression of one or more genes (and any encoded polypeptides) duringsenescence of a plant cell from a leaf, flower, fruit, or other organ orplant part with respect to the level of expression of that gene in anon-senescing, i.e., a growing or mature (but pre-senescent) cell.

In the instant description, “endogenous” refers to a molecule thatnaturally originates from within a plant, plant tissue, or plant cell.The term “endogenous polypeptide” refers to a natural or nativepolypeptide that is encoded by a plant's native gene and thus itoriginates from within the plant, plant tissue, or plant cell upon itstranslation.

A “recombinant polynucleotide” is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacids. An expression vector or cassette is an example of a “recombinantnucleic acid construct”.

A plant refers to a whole plant as well as to a plant part, such asseed, fruit, leaf, or root, plant tissue, plant cells or any other plantmaterial, e.g., a plant explant, as well as to progeny thereof, and toin vitro systems that mimic biochemical or cellular components orprocesses in a cell.

A “recombinant polypeptide” is a polypeptide produced by translation ofa recombinant polynucleotide. A “synthetic polypeptide” is a polypeptidecreated by consecutive polymerization of isolated amino acid residuesusing methods well known in the art. An “isolated polypeptide,” whethera naturally occurring or a recombinant polypeptide, is more enriched in(or out of) a cell than the polypeptide in its natural state in awild-type cell, e.g., more than about 5% enriched, more than about 10%enriched, or more than about 20%, or more than about 50%, or more,enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more,enriched relative to wild type standardized at 100%. Such an enrichmentis not the result of a natural response of a wild-type plant.Alternatively, or additionally, the isolated polypeptide is separatedfrom other cellular components with which it is typically associated,e.g., by any of the various protein purification methods herein.

“Conserved domains” are recurring units in molecular evolution, theextents of which can be determined by sequence and structure analysis. A“conserved domain” or “conserved region” as used herein refers to aregion in heterologous polynucleotide or polypeptide sequences wherethere is a relatively high degree of sequence identity between thedistinct sequences. Conserved domains contain conserved sequencepatterns or motifs that allow for their detection in, and identificationand characterization of, polypeptide sequences. A DNA-binding domain isan example of a conserved domain.

“Identity” or “similarity” refers to sequence similarity between twopolynucleotide sequences or between two polypeptide sequences, withidentity being a more strict comparison. The phrases “percent identity”and “% identity” refer to the percentage of sequence similarity found ina comparison of two or more polynucleotide sequences or two or morepolypeptide sequences. “Sequence similarity” refers to the percentsimilarity in base pair sequence (as determined by any suitable method)between two or more polynucleotide sequences. Two or more sequences canbe anywhere from 0-100% similar or identical, or any integer valuebetween 0-100%. Identity or similarity can be determined by comparing aposition in each sequence that may be aligned for purposes ofcomparison. When a position in the compared sequence is occupied by thesame nucleotide base or amino acid, then the molecules are identical atthat position. A degree of similarity or identity between polynucleotidesequences is a function of the number of identical, matching orcorresponding nucleotides at positions shared by the polynucleotidesequences. A degree of identity of polypeptide sequences is a functionof the number of identical amino acids at corresponding positions sharedby the polypeptide sequences. A degree of homology or similarity ofpolypeptide sequences is a function of the number of amino acids atcorresponding positions shared by the polypeptide sequences. Thefraction or percentage of components in common is related to thehomology or identity between the sequences. An alignment may suitably bedetermined by means of computer programs known in the art, such asMACVECTOR software, 1999 (Accelrys®, Inc., San Diego, Calif.).

“Nitrogen use efficiency (NUE)” refers to the amount of nitrogen used toproduce biomass or grain produced by a plant. Several methods have beendeveloped to estimate NUE. Depending on the plant, either biomass orgrain yield is measured. Likewise, estimates of the nitrogen used by theplant include determination of the amount of nitrogen taken up into theplant from soil or the nitrogen content of the plant.

Uptake” refers to the acquisition of nitrogen (in the form of nitrate,ammonium, or amino acids) from the soil by plant roots in plants.Generally, plants adapted to low pH and reducing soils as found inmature forests or arctic tundra tend to take up ammonium or amino acids,whereas plants adapted to higher pH and more aerobic soils prefernitrate (Maathuis, 2009, Curr. Opin. Plant Biol. 12:250-258). Uptakeoccurs at the root level primarily (although some uptake can occur onthe leaf surface in the case of atmospheric deposition) and specifictransport systems exist for nitrate, ammonium, and amino acids.

“Assimilation” refers to the reduction of nitrate to ammonium, followedby ammonium assimilation into amino acids. Nitrate reduction to nitritetakes place in both roots and shoots but is spatially separated betweenthe cytoplasm where the reduction takes place and plastids where nitritereduction to ammonium occurs Ammonium (from nitrate reduction,photorespiration or amino acid recycling) is assimilated into aminoacids via GS/GOGAT cycle. Glutamine synthetase (GS) fixes ammonium withglutamate to form glutamine. This glutamine reacts subsequently with2-oxoglutarate to form two molecules of glutamate catalyzed by theglutamine 2-oxoglutarate amino transferase (or glutamate synthase,GOGAT).

“Usage index (UI)” refers to an estimate of nitrogen use efficiency(NUE). In measuring NUE, several definitions and evaluation methods havebeen developed (Good et al., (2004) Trends Plant Sci. 9:597-605). The“usage index” factors the absolute amount of biomass produced as well asfor the ratio of biomass per unit nitrogen (tissue dry weight/nitrogencontent). A plant is considered to have a higher usage index when thesame amount of biomass is produced with less nitrogen or when morebiomass is produced with the same amount of nitrogen compared to controlplants.

“Nitrogen uptake efficiency (NupE)” is the ability of the plant toextract nitrogen from soil and fertilizer.

“Limiting nitrogen” refers to growth conditions which include a level(e.g., concentration) of nitrogen is applied which is below the levelneeded for normal plant metabolism, growth, reproduction and/orviability. Conversely, “ample nitrogen” refers to growth conditionswhich include a level (e.g., concentration) of nitrogen is applied whichin excess of the level needed for normal plant metabolism, growth,reproduction and/or viability.

A “transgenic plant” or “transformed plant” refers to a plant thatcontains genetic material not found in a wild-type plant of the samespecies, variety or cultivar. The genetic material may include anexpression vector or cassette, a transgene, an insertional mutagenesisevent (such as by transposon or T-DNA insertional mutagenesis), anactivation tagging sequence, a mutated sequence, a homologousrecombination event or a sequence modified by chimeraplasty. Typically,the foreign genetic material has been introduced into the plant by humanmanipulation, but any method can be used as one of skill in the artrecognizes.

A transgenic line or transgenic plant line refers to the progeny plantor plants deriving from the stable integration of heterologous geneticmaterial into a specific location or locations within the genome of theoriginal transformed cell.

An expression vector or cassette typically comprises apolypeptide-encoding sequence operably linked (i.e., under regulatorycontrol of) to appropriate inducible, tissue-enhanced, tissue-specific,developmentally-enhanced, or constitutive regulatory sequences thatallow for the controlled expression of the polypeptide. The expressioncassette can be introduced into a plant by transformation or by breedingafter transformation of a parent plant. A plant refers to a whole plantas well as to a plant part, such as seed, fruit, leaf, or root, planttissue, plant cells or any other plant material, e.g., a plant explant,as well as to progeny thereof, and to in vitro systems that mimicbiochemical or cellular components or processes in a cell.

It is anticipated that a transgenic or transformed plant of the instantdisclosure may have enhanced or greater nitrogen uptake relative to acontrol plant when the transgenic plant is transformed with arecombinant polynucleotide encoding any of the listed sequences, or whenthe transgenic plant contains or expresses a listed polypeptide, and asa consequence of the expression of the listed polypeptide within thetransgenic or transformed plant.

A “seed-bearing structure”, as used herein, refers to a plant part thatcomprises a developing or mature seed, and may include, but is notlimited to, an achene, berry, capsule, caryopsis or grain, circumcissilecapsule, cypsela, drupe, ear, fruit or ripened pericarp, follicle,grain, kernel, legume, loculicidal capsule, lomentum, nut, pistil, pod,poricidal capsule, samara, schizocarp, seed capsule, septicidal capsule,septifragal capsule, silicula, siliqua, silique or strobilus.

The term “overexpression” as used herein refers to a greater expressionlevel of a gene in a plant, plant cell or plant tissue, compared toexpression in a wild-type plant, cell or tissue, at any developmental ortemporal stage for the gene. Overexpression can occur when, for example,the genes encoding one or more transcription factors are under thecontrol of a strong expression signal, such as one of the promotersdescribed herein (for example, the cauliflower mosaic virus 35Stranscription initiation region). Overexpression may occur throughout aplant or in specific tissues of the plant, depending on the promoterused, as described below.

Overexpression may take place in plant cells normally lacking expressionof polypeptides functionally equivalent or identical to the presenttranscription factors. Overexpression may also occur in plant cellswhere endogenous expression of the present transcription factors orfunctionally equivalent molecules normally occurs, but such normalexpression is at a lower level. Overexpression thus results in a greaterthan normal production, or “overproduction” of the transcription factorin the plant, cell or tissue.

A “control plant” as used in the present disclosure refers to a plantsuch as a cell, seed, plant component, plant tissue, plant organ orwhole plant used to compare against an altered or experimental plantsuch as a transgenic or genetically modified plant for the purpose ofidentifying an enhanced phenotype in the altered or experimental plant.A control plant may in some cases be a transgenic plant line thatcomprises an empty vector or marker gene, but does not contain therecombinant polynucleotide of the present description that is expressedin the transgenic or genetically modified plant being evaluated. Ingeneral, a control plant is a plant of the same line or variety as theexperimental or altered plant being tested. A suitable control plantwould include a genetically unaltered or non-transgenic plant of theparental line used to generate a transgenic plant herein.

“Wild type” or “wild-type”, as used herein, refers to a plant cell,seed, plant component, plant tissue, plant organ or whole plant that hasnot been genetically modified or treated in an experimental sense.Wild-type cells, seed, components, tissue, organs or whole plants may beused as controls to compare levels of expression and the extent andnature of trait modification with cells, tissue or plants of the samespecies in which a polypeptide's expression is altered, e.g., in that ithas been knocked out, overexpressed, or ectopically expressed.

A seed-bearing structure or organ refers to a organ of a plant thatcomprises a seed such as, for example, achene, berry, capsule, caryopsisor grain, circumcissile capsule, cypsela, drupe, ear, fruit or ripenedpericarp, follicle, grain, kernel, legume, loculicidal capsule,lomentum, nut, pistil, pod, poricidal capsule, samara, schizocarp, seedcapsule, septicidal capsule, septifragal capsule, silicula, siliqua,silique, strobilus, etc.

The term “overexpression” as used herein refers to a greater expressionlevel of a gene in a plant, plant cell or plant tissue, compared toexpression of that gene in a wild-type plant, cell or tissue, at anydevelopmental or temporal stage. Overexpression can occur when, forexample, the genes encoding one or more polypeptides are under thecontrol of a strong promoter (e.g., the cauliflower mosaic virus 35Stranscription initiation region). Overexpression may also be achieved byplacing a gene of interest under the control of an inducible or tissueenhanced promoter, or may be achieved through integration of transposonsor engineered T-DNA molecules into regulatory regions of a target gene.Other means for inducing overexpression may include making targetedchanges in a gene's native promoter, e.g. through elimination ofnegative regulatory sequences or engineering positive regulatorysequences, though the use of targeted nuclease activity (such as zincfinger nucleases or TAL effector nucleases) for genome editing.Elimination of micro-RNA binding sites in a gene's transcript may alsoresult in overexpression of that gene. Additionally, a gene may beoverexpressed by creating an artificial transcriptional activatortargeted to bind specifically to its promoter sequences, comprising anengineered sequence-specific DNA binding domain such as a zinc fingerprotein or TAL effector protein fused to a transcriptional activationdomain. Thus, overexpression may occur throughout a plant, in specifictissues of the plant, or in the presence or absence of particularenvironmental signals, depending on the promoter or overexpressionapproach used.

Overexpression may take place in plant cells normally lacking expressionof polypeptides functionally equivalent or identical to the presentpolypeptides. Overexpression may also occur in plant cells whereendogenous expression of the present polypeptides or functionallyequivalent molecules normally occurs, but such normal expression is at alower level. Overexpression thus results in a greater than normalproduction, or “overproduction” of the polypeptide in the plant, cell ortissue.

“Yield” or “plant yield” refers to increased plant growth, increasedcrop growth, increased biomass, and/or increased plant productproduction (including grain), and is dependent to some extent ontemperature, plant size, organ size, planting density, light, water andnutrient availability, and how the plant copes with various stresses,such as through temperature acclimation and water or nutrient useefficiency. Increased or improved yield may be measured as increasedseed yield, increased plant product yield (plant products include, forexample, plant tissue, including ground or otherwise broken-up planttissue, and products derived from one or more types of plant tissue), orincreased vegetative yield.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Improving a plant's ability to take up and utilize nitrogen from thesoil is an important agronomic goal. Two important measures of thisability are the nitrogen uptake efficiency (NUpE), defined as the amountof nitrogen taken up per unit of plant biomass, and the Usage Index(UI), defined as plant biomass/% nitrogen. Usage index is accepted as ameasurement of nitrogen use efficiency (NUE) for vegetative stageplants. NUpE and UI are controlled both by genetic and environmentalfactors. Genetic variability in these parameters exists among cropplants. However, genetically identical plants grown on limiting nitrogenhave higher NUpE than plants grown under ample nitrogen. Improving NUpEand UI either in limiting or ample nitrogen conditions is expected toimprove crop yield. Potential strategies to improve UI or NUpE with theinstantly listed sequences or other clade member sequences includeincreased expression of regulators (or effectors) of nitrogen uptake inroots, leaves, or whole plants.

Polypeptides and Polynucleotides of the Present Description.

The present description includes increased or ectopic expression ofputative regulatory polypeptides (i.e., regulators or effectors ofnitrogen uptake) and isolated or recombinant polynucleotides encodingthe polypeptides, or novel sequence variant polypeptides orpolynucleotides encoding novel variants of polypeptides derived from thespecific sequences provided in the Sequence Listing. The polynucleotidesof the instant description may be incorporated in expression vectors forthe purpose of producing transformed plants.

Because of their relatedness at the nucleotide level, the claimedsequences will typically share at least about 40% nucleotide sequenceidentity, or at least 41%, at least 42%, at least 43%, at least 44%, atleast 45%, at least 46%, at least 47%, at least 48%, at least 49%, atleast 50%, at least 51%, at least 52%, at least 53%, at least 54%, atleast 55%, at least 56%, at least 57%, at least 58%, at least 59%, atleast 60%, at least 70%, at least 71%, at least 72%, at least 73%, atleast 74%, at least 75%, at least 76%, at least 77%, at least 78%, atleast 79%, at least 80%, at least 81%, at least 82%, at least 83%, atleast 84%, at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95% or at least 96%, at least 97%, at least 98%, atleast 99%, or about 100% sequence identity to one or more of the listedsequences or to the full-length listed sequences (e.g., any of SEQ IDNO: 2n−1, where n=1 to 1131), or to a listed sequence within or outsideof the region(s) encoding a known consensus sequence or consensusDNA-binding site, or to a listed conserved domain sequence, or within oroutside of the region(s) encoding one or all conserved domains. Thedegeneracy of the genetic code enables major variations in thenucleotide sequence of a polynucleotide while maintaining the amino acidsequence of the encoded protein.

Because of their relatedness at the protein level, the claimednucleotide sequences will typically encode a polypeptide that is atleast at least 30%, at least 31%, at least 32%, at least 33%, at least34%, at least 35%, at least 36%, at least 37%, at least 38%, at least39%, at least 40%, at least 41%, at least 42%, at least 43%, at least44%, at least 45%, at least 46%, at least 47%, at least 48%, at least49%, at least 50%, at least 51%, at least 52%, at least 53%, at least54%, at least 55%, at least 56%, at least 57%, at least 58%, at least59%, at least 60%, at least 61%, at least 62%, at least 63%, at least64%, at least 65%, at least 66%, at least 67%, at least 68%, at least69%, at least 70%, at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 90%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95% or 96%, at least 97%, at least 98%, or at least 99%,or about 100% identical in its amino acid sequence to any of, or theentire length of any of, SEQ ID NOs: 2n, where n=1 to 1131, or 2, 4, 6,8, or any even integer to 2262.

Also provided are methods for modifying yield from a plant by enhancingthe nitrogen use efficiency or a plant's nitrogen uptake of a plant bycontrolling a number of cellular processes by, for example, introducinginto a target plant a gene that encodes a polypeptide that confersenhanced nitrogen uptake. These methods are based on the ability toalter the expression of critical regulatory molecules that may beconserved between diverse plant species. Related conserved regulatorymolecules may be originally discovered in a model system such asArabidopsis and homologous, functional molecules then discovered inother plant species. The latter may then be used to confer increasedyield or photosynthetic resource use efficiency in diverse plantspecies.

Sequences in the Sequence Listing, derived from diverse plant species,may be ectopically expressed in overexpressor plants. The changes in thecharacteristic(s) or trait(s) of the plants may then be observed andfound to confer increased yield and/or increased nitrogen use efficiencyand/or nitrogen uptake. Therefore, the polynucleotides and polypeptidescan be used to improve desirable characteristics of plants.

The polynucleotides of the instant description are also ectopicallyexpressed in overexpressor plant cells and the changes in the expressionlevels of a number of genes, polynucleotides, and/or proteins of theplant cells observed. Therefore, the polynucleotides and polypeptidescan be used to change expression levels of genes, polynucleotides,and/or proteins of plants or plant cells.

The data presented herein represent the results obtained in experimentswith polynucleotides and polypeptides that may be expressed in plantsfor the purpose of increasing yield that arises from improved nitrogenuse efficiency and/or nitrogen uptake.

The polynucleotides and polypeptides of the instant description, that,when expressed in plants or crop plant have the capacity to enhancenitrogen uptake in a plant or a part of a plant relative to a controlplant or a corresponding part of the control plant, include:

SEQ ID NOs:1 and 2, AT2G24570.1 (G866) and clade member sequences SEQ IDNOs:3-74;

SEQ ID NOs:75 and 76, AT2G37430.1 (G355) and clade member sequences SEQID NOs:77-134;

SEQ ID NOs:1 and 136, AT1G07900.1 (G4083) and clade member sequences SEQID NOs:137-198;

SEQ ID NOs:199 and 200, AT1G74080.1 (G2340) and clade member sequencesSEQ ID NOs:201-204;

SEQ ID NOs:205 and 206, AT1G62300.1 (G184) and clade member sequencesSEQ ID NOs:207-286;

SEQ ID NOs:287 and 288, AT1G18860.1 (G2110) and clade member sequencesSEQ ID NOs:289-358;

SEQ ID NOs:359 and 360, AT3G23250.1 (G233) and clade member sequencesSEQ ID NOs:361-426;

SEQ ID NOs:427 and 428, AT2G26150.1 (G266) and clade member sequencesSEQ ID NOs:429-456;

SEQ ID NOs:457 and 458, AT3G15500.1 (G773) and clade member sequencesSEQ ID NOs:459-490;

SEQ ID NOs:491 and 492, AT1G16150.1 and clade member sequences SEQ IDNOs:493-538;

SEQ ID NOs:539 and 540, AT1G51800.1 and clade member sequences SEQ IDNOs:541-758;

SEQ ID NOs:759 and 760, AT1G61440.1 and clade member sequences SEQ IDNOs:761-950;

SEQ ID NOs:951 and 952, AT4G11470.1, SEQ ID NOs:953 and 954, andAT4G11480.1 and clade member sequences SEQ ID NOs:955-1212;

SEQ ID NOs:1213 and 1214, AT5G14640.1 and clade member sequences SEQ IDNOs: 1215-1374;

SEQ ID NOs:1375 and 1376, AT5G06740.1 and clade member sequences SEQ IDNOs: 1377-1400;

SEQ ID NOs:1401 and 1402, AT2G19190.1 and clade member sequences SEQ IDNOs:1403-1672;

SEQ ID NOs:1673 and 1674, AT1G57560.1 (G1319) and clade member sequencesSEQ ID NOs: 1675-1752;

SEQ ID NOs:1753 and 1754, AT2G46510.1 (G1665) and clade member sequencesSEQ ID NOs: 1755-1786;

SEQ ID NOs:1787 and 1788, AT5G54900.1 (G1940) and clade member sequencesSEQ ID NOs: 1789-1920;

SEQ ID NOs:1921 and 1922, AT3G05200.1 (G2239) and clade member sequencesSEQ ID NOs: 1923-2026;

SEQ ID NOs:2027 and 2028, AT2G34450.2 (G2898) and clade member sequencesSEQ ID NOs:2029-2054;

SEQ ID NOs:2055 and 2056, AT5G26930.1 (G348) and clade member sequencesSEQ ID NOs:2057-2090;

SEQ ID NOs:2091 and 2092, AT4G20380.1 (G347) and clade member sequencesSEQ ID NOs:2093-2166;

SEQ ID NOs:2167 and 2168, AT1G09030.1 (G486) and clade member sequencesSEQ ID NOs:2169-2242; and

SEQ ID NOs:2243 and 2244, AT2G43260.1 (G1466) and clade member sequencesSEQ ID NOs:2245-2262.

Variants of the Disclosed Sequences.

Also within the scope of the instant description is a variant of anucleic acid provided in the Sequence Listing, that is, one having asequence that differs from the one of the polynucleotide sequences inthe Sequence Listing, or a complementary sequence, that encodes afunctionally equivalent polypeptide (i.e., a polypeptide having somedegree of equivalent or similar biological activity). The variantnucleic acid may, for example, encode the same polypeptide but differ insequence from the sequence in the Sequence Listing due to degeneracy inthe genetic code. Included within this definition are polymorphisms thatmay or may not be readily detectable using a particular oligonucleotideprobe of the polynucleotide encoding polypeptide, and improper orunexpected hybridization to allelic variants, with a locus other thanthe normal chromosomal locus for the polynucleotide sequence encodingpolypeptide.

Differences between presently disclosed polypeptides and polypeptidevariants are limited so that the sequences of the former and the latterare closely similar overall and, in many regions, identical. Presentlydisclosed polypeptide sequences and similar polypeptide variants maydiffer in amino acid sequence by one or more substitutions, additions,deletions, fusions and truncations, which may be present in anycombination. These differences may produce silent changes and result infunctionally equivalent polypeptides. Thus, it will be readilyappreciated by those of skill in the art, that any of a variety ofpolynucleotide sequences is capable of encoding the polypeptides andhomolog polypeptides of the instant description. A polypeptide sequencevariant may have “conservative” changes, wherein a substituted aminoacid has similar structural or chemical properties.

Conservative substitutions include substitutions in which at least oneresidue in the amino acid sequence has been removed and a differentresidue inserted in its place. Such substitutions generally are made inaccordance with the Table 1 when it is desired to maintain the activityof the protein. Table 1 shows amino acids which can be substituted foran amino acid in a protein and which are typically regarded asconservative substitutions.

TABLE 1 Possible conservative amino acid substitutions Amino AcidConservative Residue substitutions Ala Ser Arg Lys Asn Gln; His Asp GluGln Asn Cys Ser Glu Asp Gly Pro His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr Pro Gly Ser Thr; Gly ThrSer; Val Trp Tyr Tyr Trp; Phe Val Ile; Leu

The polypeptides provided in the Sequence Listing have a novel activity,such as, for example, a regulatory activity. Although all conservativeamino acid substitutions (for example, one basic amino acid substitutedfor another basic amino acid) in a polypeptide will not necessarilyresult in the polypeptide retaining its activity, it is expected thatmany of these conservative mutations would result in the polypeptideretaining its activity. Most mutations, conservative ornon-conservative, made to a protein but outside of a conserved domainrequired for function and protein activity will not affect the activityof the protein to any great extent.

Deliberate amino acid substitutions may thus be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues, as longas a significant amount of the functional or biological activity of thepolypeptide is retained. For example, negatively charged amino acids mayinclude aspartic acid and glutamic acid, positively charged amino acidsmay include lysine and arginine, and amino acids with uncharged polarhead groups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine. More rarely, avariant may have “non-conservative” changes, e.g., replacement of aglycine with a tryptophan. Similar minor variations may also includeamino acid deletions or insertions, or both. Related polypeptides maycomprise, for example, additions and/or deletions of one or moreN-linked or 0-linked glycosylation sites, or an addition and/or adeletion of one or more cysteine residues. Guidance in determining whichand how many amino acid residues may be substituted, inserted or deletedwithout abolishing functional or biological activity may be found usingcomputer programs well known in the art, for example, DNASTAR software(see U.S. Pat. No. 5,840,544).

Conserved Domains.

Conserved domains are recurring functional and/or structural units of aprotein sequence within a protein family (for example, a family ofregulatory proteins), and distinct conserved domains have been used asbuilding blocks in molecular evolution and recombined in variousarrangements to make proteins of different protein families withdifferent functions. Conserved domains often correspond to the3-dimensional domains of proteins and contain conserved sequencepatterns or motifs, which allow for their detection in polypeptidesequences with, for example, the use of a Conserved Domain Database (forexample, at www.ncbi.nlm.nih.gov/cdd). The National Center forBiotechnology Information Conserved Domain Database defines conserveddomains as recurring units in molecular evolution, the extents of whichcan be determined by sequence and structure analysis. Conserved domainscontain conserved sequence patterns or motifs, which allow for theirdetection in polypeptide sequences (Conserved Domain Database;www.ncbi.nlm.nih.gov/Structure/cdd/cdd.shtml). A “conserved domain” or“conserved region” as used herein refers to a region in heterologouspolynucleotide or polypeptide sequences where there is a relatively highdegree of sequence identity between the distinct sequences. A ‘Myb DNAbinding domain 1’ is an example of a conserved domain.

Conserved domains may also be identified as regions or domains ofidentity to a specific consensus sequence (see, for example, Riechmannet al., 2000, Science 290, 2105-2110; Riechmann et al., 2000, Curr OpinPlant Biol 3: 423-434). Thus, by using alignment methods well known inthe art, the conserved domains of the plant polypeptides, for example,for the first or second Myb DNA binding domain proteins may bedetermined. The polypeptides in Table 2 have conserved domainsspecifically indicated by amino acid coordinate start and stop sites. Acomparison of the regions of these polypeptides allows one of skill inthe art (see, for example, Reeves and Nissen, 1990. J. Biol. Chem. 265,8573-8582; Reeves and Nissen, 1995. Prog. Cell Cycle Res. 1: 339-349) toidentify domains or conserved domains for any of the polypeptides listedor referred to in this disclosure.

Conserved domain models are generally identified with multiple sequencealignments of related proteins spanning a variety of organisms. Thesealignments reveal sequence regions containing the same, or similar,patterns of amino acids. Multiple sequence alignments, three-dimensionalstructure and three-dimensional structure superposition of conserveddomains can be used to infer sequence, structure, and functionalrelationships (Conserved Domain Database, supra). Since the presence ofa particular conserved domain within a polypeptide is highly correlatedwith an evolutionarily conserved function, a conserved domain databasemay be used to identify the amino acids in a protein sequence that areputatively involved in functions such as binding or catalysis, as mappedfrom conserved domain annotations to the query sequence. For example,the presence in a protein of a DNA binding domain that is structurallyand phylogenetically similar to one or more domains found in thesequence listing would be a strong indicator of a related function inplants (e.g., the function of regulating and/or improving nitrogen useefficiency, nitrogen uptake, and/or yield, i.e., a polypeptide with sucha domain is expected to confer enhanced nitrogen use efficiency,nitrogen uptake, and/or yield when its expression level is increasedunder the regulatory control of a tissue-enhanced promoter). Sequencesherein referred to as functionally-related and/or closely-related to thesequences or domains provided in the Sequence Listing, includingpolypeptides that are closely related to the polypeptides of the instantdescription, may have conserved domains that share at least at leastnine base pairs (bp) in length and at least 30%, at least 31%, at least32%, at least 33%, at least 34%, at least 35%, at least 36%, at least37%, at least 38%, at least 39%, at least 40%, at least 41%, at least42%, at least 43%, at least 44%, at least 45%, at least 46%, at least47%, at least 48%, at least 49%, at least 50%, at least 51%, at least52%, at least 53%, at least 54%, at least 55%, at least 56%, at least57%, at least 58%, at least 59%, at least 60%, at least 61%, at least62%, at least 63%, at least 64%, at least 65%, at least 66%, at least67%, at least 68%, at least 69%, at least 70%, at least 71%, at least72%, at least 73%, at least 74%, at least 75%, at least 76%, at least77%, at least 78%, at least 79%, at least 90%, at least 81%, at least82%, at least 83%, at least 84%, at least 85%, at least 86%, at least87%, at least 88%, at least 89%, at least 90%, at least 91%, at least92%, at least 93%, at least 94%, at least 95% or 96%, at least 97%, atleast 98%, or at least 99%, or about 100% amino acid sequence identityto the sequences provided in the Sequence Listing, and have similarfunctions in that the polypeptides of the instant description, where thepresence of the listed or claimed domains in said polypeptides ispositively correlated or associated with the function(s) of saidpolypeptides in plants. Said polypeptides may, when their expressionlevel is altered and confer at least one regulatory activity selectedfrom the group consisting of enhanced nitrogen uptake and/orassimilation, as measured by such parameters as Nitrogen UptakeEfficiency (NUpE) or Usage Index (UI), increased nitrogen useefficiency, greater yield, greater size, greater biomass, and/or greatervigor as compared to a control plant.

Methods using manual alignment of sequences similar or homologous to oneor more polynucleotide sequences or one or more polypeptides encoded bythe polynucleotide sequences may be used to identify regions ofsimilarity and conserved domains (e.g., DNA binding domains, activationdomains, localization domains, repression domains, oligomerizationdomains, or other domains that are recognizably related across plantspecies. Such manual methods are well-known of those of skill in the artand can include, for example, comparisons of tertiary structure betweena polypeptide sequence encoded by a polynucleotide that comprises aknown function and a polypeptide sequence encoded by a polynucleotidesequence that has a function not yet determined. Such examples oftertiary structure may comprise predicted α-helices, β-sheets,amphipathic helices, leucine zipper motifs, zinc finger motifs,proline-rich regions, cysteine repeat motifs, and the like.

With respect to polynucleotides encoding presently disclosedpolypeptides, a conserved domain refers to a subsequence within apolypeptide family the presence of which is correlated with at least onefunction exhibited by members of the polypeptide family, and whichexhibits a high degree of sequence homology, such as at least 30%, atleast 31%, at least 32%, at least 33%, at least 34%, at least 35%, atleast 36%, at least 37%, at least 38%, at least 39%, at least 40%, atleast 41%, at least 42%, at least 43%, at least 44%, at least 45%, atleast 46%, at least 47%, at least 48%, at least 49%, at least 50%, atleast 51%, at least 52%, at least 53%, at least 54%, at least 55%, atleast 56%, at least 57%, at least 58%, at least 59%, at least 60%, atleast 61%, at least 62%, at least 63%, at least 64%, at least 65%, atleast 66%, at least 67%, at least 68%, at least 69%, at least 70%, atleast 71%, at least 72%, at least 73%, at least 74%, at least 75%, atleast 76%, at least 77%, at least 78%, at least 79%, at least 90%, atleast 81%, at least 82%, at least 83%, at least 84%, at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95% or96%, at least 97%, at least 98%, or at least 99%, or about 100% identityto a conserved domain (e.g., any of SEQ ID NOs: 2263-2312) of apolypeptide (e.g., any of SEQ ID NOs: 2n, where n=1 to 1131) of theSequence Listing. Sequences that possess or encode for conserved domainsthat meet these criteria of percentage identity, and that havecomparable biological and regulatory activity to the present polypeptidesequences. Sequences having lesser degrees of identity but comparablebiological activity are considered to be equivalents.

Table 2 lists Arabidopsis sequence identifiers and the descriptions ofvarious domains found within the respective proteins, including thedomain names, the linear coordinates of the amino acids at the beginningand end of the respective domains, and the SEQ ID NOs: of the domainslisted in this table. It is expected that domains of clade memberpolypeptides of each of these sequences, examples of said polypeptidesbeing provided in the Sequence Listing, function similarly in plants andtheir presence is indicative of and correlated with the same functionsas the domains in the Arabidopsis sequences.

TABLE 2 Various Arabidopsis domains found in polypeptide sequences thatenhance nitrogen uptake in plants Starting Last amino amino acid acid inSEQ ID in protein protein NO: of Sequence Domain Name sequence sequenceDomain AT1G16150.1 Wall-associated receptor kinase 51 144 2263galacturonan-binding AT1G16150.1 Wall-associated kinase 174 279 2264AT1G16150.1 protein kinase catalytic (PKc) 442 708 2265 domainAT1G57560.1 Myb domain 1 (AKA SANT 14 61 2266 domain) AT1G57560.1 Mybdomain 2 (AKA SANT 67 112 2267 domain) AT1G09030.1 CCAAT-bindingtranscription 6 71 2268 factor (NF-YB) AT1G51800.1 Malectin-like 32 3532269 AT1G51800.1 Leucine rich repeat (LRR8) 412 468 2270 AT1G51800.1protein kinase catalytic (PKc) 609 855 2271 domain AT1G18860.1 WRKY DNAbinding domain 191 248 2272 AT1G61440.1 Bulb-type mannose-specific 24139 2273 lectin AT1G61440.1 S-locus glycoprotein family 198 305 2274AT1G61440.1 PAN/APPLE-like domain 322 407 2275 AT1G61440.1 Tyrosinekinase catalytic (TyrKc) 479 741 2276 domain AT1G07900.1 Lateral organboundaries (LOB) 34 133 2277 domain AT1G74080.1 SANT (aka Myb) 16 612278 AT11G74080.1 SANT (aka Myb) 69 112 2279 AT2G43260.1F-box-associated domain 102 294 2280 AT2G26150.1 HSF DNA binding domain44 136 2281 AT2G46510.1 N-terminal domain associated 48 239 2282 withbHLH-MYC domain AT2G46510.1 Helix-loop-helix domain (HLH) 394 445 2283AT2G19190.1 Malectin-like 33 359 2284 AT2G19190.1 Leucine rich repeat(LRR8) 415 474 2285 AT2G19190.1 protein kinase catalytic (PKc) 580 7662286 domain AT2G37430.1 C2H2-type zinc finger 47 72 2287 AT2G37430.1C2H2-type zinc finger 93 118 2288 AT2G24570.1 Plant zinc cluster domain191 240 2289 AT2G24570.1 WRKY DNA-binding domain 242 300 2290AT2G34450.2 High mobility group box 63 129 2291 AT3G05200.1 C3HC4 RINGfinger 125 171 2292 AT3G23250.1 SANT (aka Myb) 16 61 2293 AT3G23250.1SANT (aka Myb) 69 112 2294 AT3G15500.1 NAM 14 140 2295 AT4G22070.1 WRKY298 354 2296 AT4G11470.1 Salt stress response/antifungal 23 130 2297AT4G11470.1 Salt stress response/antifungal 189 245 2298 AT4G11470.1Tyrosine kinase catalytic (TyrKc) 342 611 2299 domain AT4G11480.1 Saltstress response/antifungal 23 126 2300 AT4G11480.1 Salt stressresponse/antifungal 142 236 2301 AT4G11480.1 Protein kinase catalytic(PKc) 325 602 2302 domain AT4G20380.1 LSD1 zinc finger 4 34 2303AT4G20380.1 LSD1 zinc finger 45 75 2304 AT4G20380.1 LSD1 zinc finger 95119 2305 AT5G54900.1 RNA recognition motif 1 61 141 2306 AT5G54900.1 RNArecognition motif 2 153 232 2307 AT5G54900.1 RNA recognition motif 3 260332 2308 AT5G06740.1 lectin domain 25 257 2309 AT5G06740.1 Proteintyrosine kinase 335 606 2310 AT5G26930.1 GATA zinc finger 28 62 2311AT5G14640.1 Protein kinase domain 77 358 2312

Orthologs and Paralogs.

Homologous sequences as described above can comprise orthologous orparalogous sequences. Several different methods are known by those ofskill in the art for identifying and defining these functionallyhomologous sequences. General methods for identifying orthologs andparalogs, including phylogenetic methods, sequence similarity andhybridization methods, are described herein; an ortholog or paralog,including equivalogs, may be identified by one or more of the methodsdescribed below.

As described by Eisen, 1998. Genome Res. 8: 163-167, evolutionaryinformation may be used to predict gene function. It is common forgroups of genes that are homologous in sequence to have diverse,although usually related, functions. However, in many cases, theidentification of homologs is not sufficient to make specificpredictions because not all homologs have the same function. Thus, aninitial analysis of functional relatedness based on sequence similarityalone may not provide one with a means to determine where similarityends and functional relatedness begins. Fortunately, it is well known inthe art that protein function can be classified using phylogeneticanalysis of gene trees combined with the corresponding species.Functional predictions can be greatly improved by focusing on how thegenes became similar in sequence (i.e., by evolutionary processes)rather than on the sequence similarity itself (Eisen, supra). In fact,many specific examples exist in which gene function has been shown tocorrelate well with gene phylogeny (Eisen, supra). Thus, “[t]he firststep in making functional predictions is the generation of aphylogenetic tree representing the evolutionary history of the gene ofinterest and its homologs. Such trees are distinct from clusters andother means of characterizing sequence similarity because they areinferred by techniques that help convert patterns of similarity intoevolutionary relationships . . . . After the gene tree is inferred,biologically determined functions of the various homologs are overlaidonto the tree. Finally, the structure of the tree and the relativephylogenetic positions of genes of different functions are used to tracethe history of functional changes, which is then used to predictfunctions of [as yet] uncharacterized genes” (Eisen, supra).

Within a single plant species, gene duplication may cause two copies ofa particular gene, giving rise to two or more genes with similarsequence and often similar function known as paralogs. A paralog istherefore a similar gene formed by duplication within the same species.Paralogs typically cluster together or in the same clade (a group ofsimilar genes) when a gene family phylogeny is analyzed using programssuch as CLUSTAL (Thompson et al., 1994, Nucleic Acids Res. 22:4673-4680; Higgins et al., 1996, Methods Enzymol. 266: 383-402). Groupsof similar genes can also be identified with pair-wise BLAST analysis(Feng and Doolittle, 1987, J. Mol. Evol. 25: 351-360). For example, aclade of very similar MADS domain transcription factors from Arabidopsisall share a common function in flowering time (Ratcliffe et al., 2001,Plant Physiol. 126: 122-132), and a group of very similar AP2 domaintranscription factors from Arabidopsis are involved in tolerance ofplants to freezing (Gilmour et al., 1998, supra). Analysis of groups ofsimilar genes with similar function that fall within one clade can yieldsub-sequences that are particular to the clade. These sub-sequences,known as consensus sequences, can not only be used to define thesequences within each clade, but define the functions of these genes;genes within a clade may contain paralogous sequences, or orthologoussequences that share the same function (see also, for example, Mount,2001, in Bioinformatics: Sequence and Genome Analysis, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., p. 543)

Regulatory polypeptide gene sequences are conserved across diverseeukaryotic species lines (Goodrich et al., 1993. Cell 75:519-530; Lin etal., 1991, Nature 353:569-571; Sadowski et al., 1988, Nature 335:563-564). Plants are no exception to this observation; diverse plantspecies possess regulatory polypeptides that have similar sequences andfunctions. Speciation, the production of new species from a parentalspecies, gives rise to two or more genes with similar sequence andsimilar function. These genes, termed orthologs, often have an identicalfunction within their host plants and are often interchangeable betweenspecies without losing function. Because plants have common ancestors,many genes in any plant species will have a corresponding orthologousgene in another plant species. Once a phylogenic tree for a gene familyof one species has been constructed using a program such as CLUSTAL(Thompson et al., 1994, supra; Higgins et al., 1996, supra) potentialorthologous sequences can be placed into the phylogenetic tree and theirrelationship to genes from the species of interest can be determined.Orthologous sequences can also be identified by a reciprocal BLASTstrategy. Once an orthologous sequence has been identified, the functionof the ortholog can be deduced from the identified function of thereference sequence.

The polypeptides sequences belong to distinct clades of polypeptidesthat include members from diverse species. In each case, most or all ofthe clade member sequences derived from both eudicots and monocots havebeen shown and are predicted to enhance nitrogen uptake to conferincreased yield when the sequences were overexpressed under theregulatory control of, for example, a root, root cap, root meristem,root vasculature, vascular, and/or green tissue-enhanced promoter. Thesestudies each demonstrate that evolutionarily conserved genes fromdiverse species are likely to function similarly (i.e., by regulatingsimilar target sequences and controlling the same traits), and thatpolynucleotides from one species may be transformed into closely-relatedor distantly-related plant species to confer or improve traits.

Orthologs and paralogs of presently disclosed polypeptides may be clonedusing compositions provided by the present description according tomethods well known in the art. cDNAs can be cloned using mRNA from aplant cell or tissue that expresses one of the present sequences.Appropriate mRNA sources may be identified by interrogating Northernblots with probes designed from the present sequences, after which alibrary is prepared from the mRNA obtained from a positive cell ortissue. Polypeptide-encoding cDNA is then isolated using, for example,PCR, using primers designed from a presently disclosed gene sequence, orby probing with a partial or complete cDNA or with one or more sets ofdegenerate probes based on the disclosed sequences. The cDNA library maybe used to transform plant cells. Expression of the cDNAs of interest isdetected using, for example, microarrays, Northern blots, quantitativePCR, or any other technique for monitoring changes in expression.Genomic clones may be isolated using similar techniques to those.

Examples of orthologs of the Arabidopsis polypeptide sequences and theirfunctionally similar orthologs are listed in the present SequenceListing. In addition to the Arabidopsis polypeptide sequences in theSequence Listing, these orthologs are phylogenetically and structurallysimilar to the sequences in the Sequence Listing and can also functionin a plant by increasing nitrogen use efficiency, nitrogen uptake,yield, vigor, and/or biomass when ectopically and preferentiallyexpressed in a plant or in a plant organ. Since a significant number ofthese sequences are phylogenetically and sequentially related to eachother and may be shown to increase yield from a plant and/or nitrogenuptake, one skilled in the art would predict that other similar,phylogenetically related sequences, including those falling within thepresent clades of polypeptides or having the same consensus sequences orwhich are sequentially similar, having a disclosed minimum percentageidentity to one another or the listed Arabidopsis polypeptide, wouldalso perform similar functions when ectopically expressed under theregulatory control of the disclosed promoters or other root, root cap,root meristem, root vasculature, vascular, and/or green tissue-enhancedpromoters.

Promoters.

The tissue-enhanced promoters in Table 3 preferentially regulate geneexpression in root, root cap, root meristem, root vasculature, and/orvascular tissue structures or organs relative to other tissues in aplant. Examples of tissue-enhanced promoters may also be found in theSequence Listing as SEQ ID NOs: 2313 to 2349.

The choice of promoter may also include a constitutive promoter or apromoter with enhanced activity in a tissue capable of photosynthesis(also referred to herein as a “green tissue promoter”, a “photosyntheticpromoter” or a “photosynthetic tissue-enhanced promoter”) such as a leaftissue or other green tissue. Examples of photosynthetic or green tissuepromoters include for example, an RBCS3 promoter (SEQ ID NO: 2323), anRBCS4 promoter (SEQ ID NO: 2324) others such as the At4g01060 promoter(SEQ ID NO: 2325), the latter regulating expression in a guard cell, orrice sequences SEQ ID NOs: 2326 to 2349, shown in Table 3 or in theSequence Listing.

TABLE 3 Exemplary promoters SEQ Organism from ID which promoter PromoterNO is derived Tissue Reference Os03g01700 2350 rice root Li et al.,2013. Plant Sci. 207:37-44 Os02g37190 2351 rice root Li et al., 2013.Plant Sci. 207:37-44 rolD 2352 Agrobacterium root Leach and Aoyagi.1991. Plant Sci. 79:69- rhizogenes 76 RCc3 2353 rice root Xu et al.1995. Plant Mol. Biol. 27:237-248 TobRB7 2354 tobacco root Yamamoto,etal. 1991. Plant Cell 3:371-382 Pyk10 2355 Arabidopsis root Nitz et al.2001. Plant Sci. 161:337-346 PmPR10-1.14 Pinus monticola root Liu andEkramoddoullah. 2003. Plant Mol. Biol. 52:103-120 HvPht1;1 2356 barleyroot Schünmann et al. 2004. J. Exp. Bot. 55:855-865 HvPht1;2 2357 barleyroot Schünmann 2004. J. Exp. Bot. 55:855-865 MsPRP2 alfalfa root Winicovet al. 2004. Planta 219:925-935 PHT1 2358 Arabidopsis root Koyama et al.2005. J. Biosci. Bioeng. 99: 38-42 At1g73160 2359 Arabidopsis rootVijaybhaskar et al. 2008. J. Biosci. 33; 185- 193 AtCel5 2360Arabidopsis root cap del Campillo et al. 2004. Plant Mol. Biol. 56:309-323 ARSK1 2361 Arabidopsis root Hwang and Goodman. 1995. Plant J.8:37- 43 RSI1 2362 tomato root Taylor and Scheuring 1994. Mol. Gen.meristem and Genet. 243:148-157 vasculature AT2G39850 2313 Arabidopsisvascular SEQ ID NO: 27 of US patent pub. US20110179520 AT2G03500 2314Arabidopsis vascular SEQ ID NO: 22 US patent pub. US20110179520AT3G42670 2315 Arabidopsis vascular SEQ ID NO: 34 US patent pub.US20110179520 AT1G24735 2316 Arabidopsis vascular SEQ ID NO: 5 US patentpub. U520110179520 AT5G56530 2317 Arabidopsis vascular SEQ ID NO: 63 USpatent pub. US20110179520 AT3G12750 2318 Arabidopsis vascular SEQ ID NO:29 US patent pub. U520110179520 AT3G16340 2319 Arabidopsis vascular SEQID NO: 31 US patent pub. US20110179520 AT1G65150 2320 Arabidopsisvascular SEQ ID NO: 16 US patent pub. U520110179520 AT5G27690 2321Arabidopsis vascular SEQ ID NO: 58 US patent pub. U520110179520AT1G10155 2322 Arabidopsis vascular SEQ ID NO: 2 US patent pub.U520110179520 SUC2 2363 Arabidopsis vascular Truernit and Sauer. 1995.Planta 196:564- 570 CoYMV Commelina vascular Medberry et al. 1992. PlantCell 4:185-192 yellow mottle virus Sucrose Rice vascular Wang et al.1992. Plant Mol. Biol. 19:881- synthase 885 Sucrose 2364 maize vascularYang and Russell. 1980. Proc. Natl Acad. synthase Sci. USA 87:4144-4148GmSBP2 2365 Glycine max vascular Waclawovsky et al. 2006. BBA-GeneStruct. Expr. 1759:89-98 Rplec2 2366 Robinia vascular Yoshida et al.2002. J. Plant Physiol. pseudoacacia 159:757-764 PP2 Cucurbita vascularJiang et al. 1999. J. Agric. Biotechnol. 7:63- maxima 68 PP2 Cucurbitavascular Thompson and Larkins. 1996. US patent maxima pub. U5005495007ArolC 2367 Agrobacterium vascular Schmulling et al. 1989. Plant Cell1:665- rhizogenes 670 RTBV rice tungro vascular Yin and Beachy. 1997.Plant J. 12:1179- bacilliform virus 1188 DX1 2368 rice green tissue Zhouand Lin. 2012. Plant Cell Rep. (Os12g33120) 31:1159-1172.doi:10.1007/s00299-012- 1238-8 RBCS3 2323 tomato green tissue Wanner andGruissem. 1991. Plant Cell 3: 1289-1303 RBCS4/RBCS1A 2324 Arabidopsisgreen tissue PEPC 2369 maize green tissue Koziel et al. 1993. NatureBiotechnol. 11:194-200 prG682 2325 Arabidopsis guard cell At4g01060Os02g09720 2326 rice green tissue Os05g34510 2327 rice green tissueOs11g08230 2328 rice green tissue Os01g64390 2329 rice green tissueOs06g15760 2330 rice green tissue Os12g37560 2331 rice green tissueOs03g17420 2332 rice green tissue Os04g51000 2333 rice green tissueOs01g01960 2334 rice green tissue Os05g04990 2335 rice green tissueOs02g44970 2336 rice green tissue Os01g25530 2337 rice green tissueOs03g30650 2338 rice green tissue Os01g64910 2339 rice green tissueOs07g26810 2340 rice green tissue Os07g26820 2341 rice green tissueOs09g11220 2342 rice green tissue Os04g21800 2343 rice green tissueOs10g23840 2344 rice green tissue Os08g13850 2345 rice green tissueOs12g42980 2346 rice green tissue Os03g29280 2347 rice green tissueOs03g20650 2348 rice green tissue Os06g43920 2349 rice green tissuepsaDb tobacco green tissue Yamamoto et al. 1997. Plant J. 12:255-265gapb 2370 Arabidopsis green tissue Kwon et al. 1994. Plant Physiol.105:357- 367 cab 1 wheat green tissue Gotor et al. 1993. Plant J.3:509-518 cab 6 pine green tissue Yamamoto et al. 1994. Plant CellPhysiol. 35:773-778 rbcs activase spinach green tissue Orozco et al.1993. Plant Mol. Biol. 23: 1129-1138 ppdk 2371 maize green tissueMatsuoka et al. 1993. Proc. Natl. Acad. Sci. USA 90:9586-9590 lhcb1 * 22372 tobacco green tissue Cerdan et al. 1997. Plant Mol. Biol.33:245-255

EXAMPLES

It is to be understood that this description is not limited to theparticular devices, machines, materials and methods described. Althoughparticular embodiments are described, equivalent embodiments may be usedto practice the claims.

The specification, now being generally described, will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present description and are not intended to limit the claims ordescription. It will be recognized by one of skill in the art that apolypeptide that is associated with a particular first trait may also beassociated with at least one other, unrelated and inherent second traitwhich was not predicted by the first trait.

Example I. Identification of Regulators of UI and NUpE Through GeneExpression Profiling

Plants grown under limiting nitrogen conditions take up and utilizenitrogen more efficiently than do plants grown under ample nitrogen. Toelucidate the regulatory networks controlling these differences, atranscriptional profiling experiment was performed on Arabidopsis plantsgrown under conditions of limiting (2 mM) and ample (10 mM) nitrogen.Leaf and silique tissue were harvested beginning in the vegetative phaseand continuing through seed development to create a developmental timeseries under these two different nutrient regimes.

Plant Growth and Tissue Isolation.

Plants were grown in pots containing three volumes of fritted clay atbottom and one volume of fine sand on top. Pots were pre-soaked innutrient solution containing either 2 mM (low N) or 10 mM (high N)nitrate solution. Phosphate (0.25 mM), sulfate (0.25 mM), magnesium(0.25 mM), and sodium (0.20 mM) were present in both solutions at thesame concentration. The difference between low and high N solutionsaffects only potassium (5.25 mM and 2.75 mM in high and low N solutions,respectively), calcium (2.50 mM and 0.50 mM, respectively), and chlorideions (0.25 mM and 0.70 mM, respectively). Pots were placed in Convirongrowth chambers at a day temperature of 22° C. (19° C. night) with a 16hr photoperiod at an initial light intensity of ˜100 μmol m−2 s−1 and afinal light intensity of −140 μmol m−2 s−1 at plant height. After twoweeks, flats were moved to a commercial ebb and flow hydroponic system(Bigfoot, American Hydroponics, Arcata, Calif.) in an Enconair growthchamber with a 16 hr photoperiod at a light intensity of 140 μmol m−2s−1 at plant height, and a 22° C. day temperature (19° C. night). Plantswere supplied with nutrient solution containing 2 mM or 10 mM nitratethrough a pumping system once every 8 hrs.

Leaf 7 (the seventh leaf formed by each plant) was tagged with thread 21days after sowing (“days after sowing” abbreviation: DAS). Collection ofleaf 7 was started at 22 DAS and continued every other day until 42 DAS,for a total of 11 time points. Sampling of siliques started whensiliques reached stage 16 (floral organs withering) and 2, 4, 6, and 8days post stage 16, for a total of five time points. At each time point,leaf 7 and siliques were harvested from 10-12 plants and eight plants,respectively, with plants being selected to minimize any potentialeffects of position within the hydroponic tub and growth room.

RNA Isolation.

Leaves were pulverized in liquid nitrogen with a mortar and pestle.Approximately 100 μl of frozen tissue was combined with buffer RA1 with1% β-mercaptoethanol (NucleoSpin® 96 RNA, Macherey-Nagel, Bethlehem,Pa.), and total RNA was isolated according to the manufacturer'sspecifications and implemented as a custom method on a Biomek® FX^(P)(Beckman Coulter, Brea, Calif.) liquid handling workstation. In somecases, RNA yields were too low to satisfy the target of 2 μg for makingadapter libraries. In these cases a second aliquot of 100 μL tissue wastaken to perform an additional RNA extraction. RNA from the secondextraction was used if the yield was sufficient. Otherwise, RNA waspooled from both extractions.

Silique RNA was extracted following a modified protocol (Meng andFeldman, 2010, Biotechnol. J. 5: 183-186). This procedure employs amodified, high pH (pH 9.5) extraction buffer. An RNAeasy® kit (Quiagen,Hilden, Germany) was subsequently used to purify the RNA.

Sequence Library Creation.

The starting total RNA concentration was measured using a NanoDrop®Spectrophotometer (Thermo Scientific™, Waltham, Mass.) and 2 μg of totalRNA was used as the entry point to the TruSeq RNA Sample Prep method(15008136_A, Illumina®, San Diego, Calif.). All steps were performedaccording to manufacturer specifications as indicated in theHigh-Throughput (HT) Protocol, but implemented as a custom method on aBiomek FX^(P) liquid handling workstation. In brief, poly-A containingmRNA molecules were purified using poly-T oligo-attached magnetic beads(2-rounds), fragmented with heat to a size of 120-200 bp, and thenreverse transcribed using random hexamer primers. Double stranded cDNAwas produced and indexed adapters were attached by ligation (Illumina,Inc.). Library size distribution was assessed by capillary gelelectrophoresis, and then normalized based on quantification based onabsorbance, fluorescence and quantitative polymerase chain reaction(qPCR) using primers targeting the adapter sequences.

Sequencing.

Normalized libraries were pooled proportionally based on the results ofthe RT-PCR quantification and prepared for sequencing. Cluster formationon the flowcell was performed with the Illumina Cluster Station systemand the TruSeq SR Cluster Kit v5 (Illumina). Flowcells were sequenced onthe Genome Analyzer IIx (GAIIx) system using TruSeq SBS Kit v5(Illumina) to produce single end 29 bp reads, plus a 7-cycle index read.At least 15 million reads per sample was acquired.

Data Processing.

Output from the GAIIx was pre-processed using Illumina's CASAVA softwarev1.8.1 to produce one file of short-read basecall profiles for eachsample. Short reads in basecall profiles were aligned to transcriptelements (N=41671) in the genomic reference Arabidopsis transcriptome(TAIR10_GFF3_genes.gff) using the TopHat program v2.0.4 with defaultparameters. Resulting TopHat alignment profiles were converted to SAMformat using SAMTools v0.1.18. For each sample, absolute expressionprofiles were quantified using HTSeq v0.5.3p9 to produce counts for each“gene”, aggregating across all aligned transcripts of the gene asstructurally defined in the GFF. HTSeq was run in “union” mode innon-strand specific fashion. Gene expression profiles were then analyzedwith EdgeR v3.0 to account for batch and treatment effects. Only genesthat had more than two reads per million total reads in three or moresamples were modeled.

Contrasts were created to compare the combinations of experimentalfactors against appropriate controls using a log-likelihood ratio testwith p-values calculated using a χ2 distribution. Since these analysesproduce results using many statistical tests, a Benjamini-Hochberg falsediscovery rate (FDR) multiple test correction was applied to p-valueswithin each profile. To be considered differentially expressed betweenexperimental conditions, a given gene was required to show at least a1.3 fold difference of expression with a statistical significance ofp-FDR<0.05.

Selection of Genes.

The transcript profiles of leaves at each of the 11 time points (22, 24,26, 28, 30, 32, 34, 36, 38, 40 and 42 DAS) and silique (stage 16, 2, 4,6 and 8 days post stage 16) were obtained, comparisons were made (i)between plants supplied with low and high nitrate at matched time pointand (ii) against the initial time point (22 DAS and stage 16 for leafand silique, respectively). The expression profiles of the low N andhigh N grown plants were relatively similar until 30 DAS, correspondingto the time when seed filling began, when a large number of genes beganto be differentially expressed in low N conditions and visual evidenceof senescence initiation was first noted in leaf 7 in low N. Theresulting data were analyzed through a number of computationalapproaches, e.g., SplineCluster (Heard et al., 2006, J. Am. Statist.Assoc. 101: 18-29; Heard 2011, J. Comput. Graph. Stat. 20: 920-936,Cornet (cornet.psb.ugent.be/), Gene Ontology analysis and variousnetwork inference approaches) to identify potential regulators of NUEand NUpE. Differentially expressed regulatory genes (transcriptionfactors, kinases, phosphatases, RING ubiquitin ligases) were alsoindividually examined. Two types of genes were selected for experimentalanalysis:

1) Regulatory genes consistently more highly expressed in leaves ofplants grown in low N than in leaves of plants grown in high N before 30DAS. These are candidate genes for up-regulation in leaves, roots,vascular tissue, or whole plants to improve nitrogen uptake and/orassimilation.

2) Regulatory genes consistently more highly expressed in leaves ofplants grown in high N than in leaves of plants grown in low N before 30DAS. These are candidate genes for down-regulation leaves, roots,vascular tissue, or whole plants to improve nitrogen uptake and/orassimilation.

Example II. Identification of Regulators of UI and NUpE Through WholePlant Soil-Based or Plate-Based Screening Assays

Plants grown in plate-based studies or under normal growth conditions insoil may exhibit characteristics of enhanced nitrogen uptake efficiencyor nitrogen use efficiency. For example, plants overexpressing aparticular gene that are larger or darker green compared to controlplants may be able to utilize nitrogen more effectively. In this way,large numbers of genes may be rapidly screened by examining changes inbiomass, chlorophyll and nitrogen content.

Plant Growth Conditions for Plate-Based Assays.

Seeds from Arabidopsis lines overexpressing various genes were chlorinegas sterilized and stratified at 4° C. for three days and spread ontoplates containing a sucrose-based media augmented with vitamins (80%MS+Vit, 1% sucrose, 0.65% Phytoblend™ agar) and kanamycin (3.5 mg/L).The media on these plates can be amended by altering the sucrose ornitrogen levels to alter seedling root and shoot development (forexample, see Malamy and Ryan, 2001, Plant Physiol. 127: 899-909, Zhangand Forde, 2000, J. Exp. Bot. 51:51-59, Watch-Liu et al., 2006, Ann.Bot. 97: 875-881, Gifford et al., 2013, PLoS Genet. 9(9): e1003760.doi:10.1371/journal.pgen.1003760). The effect of the gene beingoverexpressed may be determined by growing control and overexpressinglines on the same plate and observing if root and shoot architecture(e.g. root length, root branching, dark green rosette leaves) arealtered.

Plant Growth Conditions for Soil Based Experiments.

Seeds from Arabidopsis lines overexpressing various genes were chlorinegas sterilized using a standard protocol and spread onto platescontaining a sucrose-based media augmented with vitamins (80% MS+Vit, 1%sucrose, 0.65% PhBL) and either kanamycin (80% MS+Vit, 1% sucrose, 0.65%Phytoblend agar, kanamycin 3.5 mg/L) or sulfonamide (80% MS+Vit, 1%sucrose, 0.65% Phytoblend agar, asulam (1.5 mg/L) where selection wasrequired. Seeds were stratified in the dark on plates, at 4° C. forthree days, then moved to a walk-in growth chamber (Conviron MTW120,Conviron Controlled Environments Ltd, Winnipeg, Manitoba, Canada)running at a 10 h photoperiod at a photosynthetic photon flux of ca. 200μmol m⁻² s⁻¹ at plant height and a photoperiod/night temperature regimeof 22° C./19° C. After seven days of light exposure, seedlings weretransplanted into 164 ml volume pots containing autoclaved ProMix® soilaugmented with beneficial organisms for pest and disease control. Allpots were returned to the same growth-chamber and were left standing inwater, covered with a lid for the first seven days. This protocol keptthe soil moist during this period. Seven days after transplanting, lidswere removed and a watering and nutrition regime was begun. All plantsreceived a fertilizer treatment once a week (80% Peter's NPKfertilizer), and water on two other days.

Rosette Biomass and Chemical Analysis for Soil Based Experiments.

The dry weight of whole Arabidopsis rosettes was measured after beingdried down at 80° C. for 24 hours, a time found to be sufficient toreach constant weight. Samples were taken after 35 to 38 days growth,and used as an assay of aboveground productivity at growth light.Typically, five replicate rosettes were sampled per Arabidopsis linebeing screened. After weighing, the five rosettes sampled for each linescreened were pooled together and ground to a fine powder. The pooledsample generated was sub-sampled and ca. 4 μg samples were analyzed forC and N using an elemental analyzer.

Example III. Production of Transgenic Plants

The above-identified regulatory genes may combined with tissue-specificor constitutive promoters and be used to create constructs to transformplants. Transformed plants may be prepared using the following methods,although these examples are not intended to limit the description orclaims.

Transformation.

Transformation of Arabidopsis is typically performed by anAgrobacterium-mediated protocol based on the method of Bechtold andPelletier, 1998, Methods Mol. Biol. 82:259-266.

Plant Preparation.

Arabidopsis seeds are sown on mesh covered pots. The seedlings arethinned so that 6-10 evenly spaced plants remain on each pot 10 daysafter planting. The primary bolts are cut off a week beforetransformation to break apical dominance and encourage axillary shootsto form. Transformation is typically performed at 4-5 weeks aftersowing.

Bacterial Culture Preparation.

Agrobacterium stocks are inoculated from single colony plates or fromglycerol stocks and grown with the appropriate antibiotics and grownuntil saturation. On the morning of transformation, the saturatedcultures are centrifuged and bacterial pellets are re-suspended inInfiltration Media (0.5×MS, 1×B5 Vitamins, 5% sucrose, 1 mg/mlbenzylaminopurine riboside, 200 μl/L Silwet L77) until an A600 readingof 0.8 is reached.

Transformation and Seed Harvest.

The Agrobacterium solution is poured into dipping containers. All flowerbuds and rosette leaves of the plants are immersed in this solution for30 seconds. The plants are laid on their side and wrapped to keep thehumidity high. The plants are kept this way overnight at 22° C. and thenthe pots are unwrapped, turned upright, and moved to the growth racks.

The plants are maintained on the growth rack under 24-hour light untilseeds are ready to be harvested. Seeds are harvested when 80% of thesiliques of the transformed plants are ripe (approximately five weeksafter the initial transformation). This seed is deemed T0 seed, since itis obtained from the T0 generation, and is later plated on selectionplates (kanamycin, sulfonamide or glyphosate). Resistant plants that areidentified on such selection plates comprise the T1 generation.

For polynucleotides encoding polypeptides used in these experiments,RT-PCR may be performed to confirm the ability of cloned promoterfragments to drive expression of the polypeptide transgene in plantstransformed with the vectors.

T1 plants transformed with promoter-TF combinations comprised within anucleic acid construct are subjected to morphological analysis.Promoters that produce a substantial amelioration of the negativeeffects of TF overexpression are subjected to further analysis bypropagation into the T2 generation, where the plants are analyzed for analtered trait relative to a control plant.

Example IV. Evaluating Transgenic Arabidopsis Usage Index (UI) andNitrogen Uptake Efficiency (NupE) Under Limiting or Ample NitrogenConditions Using Whole Plant Assays

Plant Growth and Tissue Isolation.

Transgenic seeds were surface sterilized using chlorine gas, plated onselective media, and stratified for three days at 4° C. in the dark.Plates were incubated at 22° C. under a light intensity of approximately100 μmole m⁻² sec⁻¹ for seven days (Conviron ATV-26 growth chamber)under a L:D 10:14 regime. Seedlings were then transplanted into squarepots (60 mm×60 mm) containing fritted clay topped with a small (10 mm)layer of medium particle sized sand and kept covered with a plastic domefor another seven days to maintain humidity while they becameestablished. Plants are grown in this soil mixture under fluorescentlights, at a light intensity of approximately 100 μmole m⁻² sec⁻¹ and atemperature of 22° C. (L:D 10:14). Plants were cultivated under limitingnitrogen (2 mM nitrate) or ample nitrogen (10 mM nitrate) conditions.Phosphate (0.25 mM), sulfate (0.25 mM), magnesium (0.25 mM), and sodium(0.20 mM) were present in both solutions at the same concentration. Thedifference between low and high N solutions affects only potassium (5.25mM and 2.75 mM in high and low N solutions, respectively), calcium (2.50mM and 0.50 mM, respectively), and chloride ions (0.25 mM and 0.70 mM,respectively). Pots were watered three times per daily every eight husing a commercial ebb and flow hydroponic system (Bigfoot, AmericanHydroponics, Arcata Calif.).

¹⁵N Labeling and Harvest.

¹⁵N uptake was estimated 32 days after sowing when plants were stillvegetative. The unlabelled watering solution was replaced by a¹⁵N-containing solution that had the same nutrient composition exceptthat ¹⁴NO₃ was replaced by ¹⁵NO₃ at 2.5% enrichment. All pots werewatered for 24 h by immersing the base of the pot with a volume oflabeled solutions sufficient to cover the lower 35 mm of the pot. After24 h, the rosette was separated from its root to stop ¹⁵N uptake.Rosettes were then dried and their dry weight was determined. Four tosix replicates were harvested for uptake and remobilization experiments.

Determination of Total Nitrogen Content and ¹⁵N Abundance.

After drying and weighing each sample, material was ground in a beadmill to obtain a homogenous fine powder. A subsample of 2 to 3 mg wasweighed into tin capsules to determine the total N content and ¹⁵Nabundance at the Stable Isotope Facility at UC Davis. Samples wereanalyzed for ¹⁵N isotopes using a PDZ Europa ANCA-GSL elemental analyzerinterfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (SerconLtd., Cheshire, UK). Samples were combusted at 1000° C. in a reactorpacked with chromium oxide and silvered cobaltous/cobaltic oxide.Following combustion, oxides were removed in a reduction reactor(reduced copper at 650° C.). The helium carrier then flowed through awater trap (magnesium perchlorate). N₂ and CO₂ were resolved on aCarbosieve® GC column (65° C., 65 mL/min) before entering the IRMS.During analysis, samples were interspersed with several replicates of atleast two different laboratory standards. These laboratory standards(selected to be compositionally similar to the samples being analyzed)were previously calibrated against NIST Standard Reference Materials(IAEA-N1, IAEA-N2, IAEA-N3, USGS-40, and USGS-41). A sample'spreliminary isotope ratio is measured relative to reference gasesanalyzed with each sample. These preliminary values are finalized bycorrecting the values for the entire batch based on the known values ofthe included laboratory standards. The long term standard deviation is0.3 per mil for ¹⁵N. The ¹⁵N abundance was calculated as atom percent (A%=(¹⁵N)/(¹⁵N+¹⁴N)) and for unlabeled plant controls (A %_(control)) was0.3660. The ¹⁵N enrichment (E %) of the plant material was then definedas E %=A %_(sampie)−A %_(control).

Determination of Nitrogen Related Parameters (Nitrogen Usage Index andNitrogen Uptake Efficiency).

A number of traits related to nitrogen uptake and remobilization can bederived from tissue dry weight, tissue % nitrogen (% N), and tissue E %.

Usage Index (UI)

Usage index (UI)=tissue dry weight/N %

N uptake efficiency (NupE)

NupE=¹⁵N/tissue dry weight=E %×N %×100

Example V. Experimental Results

This Example provides experimental observations for transgenic plantsoverexpressing GATA23, MYB50, AT2G43260, RBP45A, AtbHLH017, NF-YB4,ATL6, LSD1, WRKY17, ZAT11 and HMGB14, and results observed for improvednitrogen uptake. Two or three independent lines for each gene wereevaluated for Usage Index (UI) and N uptake efficiency (NupE) underconditions of limiting and ample nitrogen as described above.

GATA23:

One GATA23 line showed a statistically significant increase in NupE whenevaluated under limiting N conditions (150% of control). Two independentlines showed an increase in UI under ample N conditions (360% and 240%of control), including the same line that showed an improvement in NupEunder limiting N conditions.

TABLE 4 Enhanced nitrogen uptake in plants ectopically expressing GATA23Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incr Limitingnitrogen GATA23 1 72.0 — 0.18 — GATA23 2 72.4 — 0.21 — GATA23 3 88.40.087 150 0.20 — Control 59.0 0.16 Ample nitrogen GATA23 1 55.7 — 0.250.000 360 GATA23 2 55.5 — 0.17 GATA23 3 60.6 — 0.17 0.098 240 Control56.9 0.07

MYB50.

One MYB50 line showed a statistically significant increase in NupE whenevaluated under ample N conditions. Two independent MYB50 lines showedan increase in UI under limiting N conditions, and all three linestested showed an improvement in UI under ample N conditions.

TABLE 5 Enhanced nitrogen uptake in plants ectopically expressing MYB50Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incr Limitingnitrogen MYB50 1 62.8 — 0.26 0.054 163 MYB50 2 76.5 — 0.25 — MYB50 377.5 — 0.27 0.010 169 Control 59.0 0.16 Ample nitrogen MYB50 1 62.3 —0.30 0.000 429 MYB50 2 109.3 0.007 192 0.21 0.007 300 MYB503 3 98.4 —0.28 0.090 400 Control 56.9 0.07

AT2G43260:

Two independent AT2G43260_lines showed an increase in UI under limitingN conditions, and all three lines tested showed an improvement in UIunder ample N conditions.

TABLE 6 Enhanced nitrogen uptake in plants ectopically expressingAT2G43260 Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incrLimiting nitrogen AT2G43260 1 64.7 — 0.27 0.034 168 AT2G43260 2 73.2 —0.23 — AT2G43260 3 72.5 — 0.33 0.000 206 Control 59.0 0.16 Amplenitrogen AT2G43260 1 64.7 — 0.18 0.034 257 AT2G43260 2 68.7 — 0.18 0.052257 AT2G43260 3 62.5 — 0.18 0.067 257 Control 56.9 0.07

RBP45A:

All three independent RBP45A lines tested showed a significant increasein UI when grown under limiting N conditions, and one of these linesalso showed a significant increase in UI under ample N conditions.

TABLE 7 Enhanced nitrogen uptake in plants ectopically expressing RBP45AGene Event NupE UI Name # Ave. p val % incr Ave. p val % incr Limitingnitrogen RBP45A 1 68.8 — 0.36 0.000 225 RBP45A 2 85.4 — 0.27 0.012 169RBP45A 3 71.4 — 0.26 0.031 162 Control 59.0 0.16 Ample nitrogen RBP45A 163.5 — 0.16 — RBP45A 2 65.1 — 0.18 0.036 257 RBP45A 3 80.9 — 0.12 —Control 56.9 0.07

AtbHLH017:

All three independent AtbHLH017 lines tested showed a significantincrease in UI when grown under limiting N conditions.

TABLE 8 Enhanced nitrogen uptake in plants ectopically expressingAtbHLH017 Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incrLimiting nitrogen AtbHLH017 1 65.5 — 0.27 0.027 169 AtbHLH017 2 57.8 —0.27 0.017 169 AtbHLH017 3 65.2 — 0.28 0.008 175 Control 59.0 0.16 Amplenitrogen AtbHLH017 1 70.8 — 0.14 — AtbHLH017 2 61.7 — 0.12 — AtbHLH017 382.8 — 0.14 — Control 56.9 0.07

NF-YB4:

One NF-YB4 line showed a significant increase in NupE when grown underample N.

TABLE 9 Enhanced nitrogen uptake in plants ectopically expressing NFYB4Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incr Limitingnitrogen NF-YB4 1 51.2 — 0.13 — NF-YB4 2 50.2 — 0.08 — NF-YB4 3 52.5 —0.09 — Control 59.0 0.16 Ample nitrogen NF-YB4 1 72.7 — 0.09 — NF-YB4 266.4 — 0.08 — NF-YB4 3 98.4 0.058 172 0.09 — Control 56.9 0.07

ATL6:

Two independent ATL6 lines showed an increase in NupE under limiting N,and in UI under ample N conditions.

TABLE 10 Enhanced nitrogen uptake in plants ectopically expressing ATL6Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incr Limitingnitrogen ATL6 1 93.3 0.026 158 0.22 — ATL6 2 99.2 0.005 168 0.24 —Control 59.0 .16 Ample nitrogen ATL6 1 89.2 — 0.23 0.001 328 ATL6 2 78.0— 0.25 0.000 357 Control 56.9 0.07

LSD1:

Two independent LSD1 overexpressing lines showed an increase in NupEunder both limiting and ample N conditions. One line also showed anincrease in UI under limiting N.

TABLE 11 Enhanced nitrogen uptake in plants ectopically expressing LSD1Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incr Limitingnitrogen LSD1 1 97.7 0.002 152 0.13 — LSD1 2 111.0 0.000 173 0.23 0.000176 Control 64.0 0.13 Ample nitrogen LSD1 1 118.8 0.008 152 0.19 — LSD12 110.2 0.091 142 0.20 — Control 77.6 0.12

WRKY17:

Three independent WRKY17 overexpressing lines showed a significantincrease in NupE under limiting N conditions, and two of these linesalso showed a significant increase under ample N. All three lines alsoshowed an increase in UI under both limiting and ample N.

TABLE 12 Enhanced nitrogen uptake in plants ectopically expressingWRKY17 Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incrLimiting nitrogen WRKY17 1 100.3 0.000 157 0.30 0.000 231 WRKY17 2 118.60.000 185 0.30 0.000 231 WRKY17 3 110.9 0.000 173 0.33 0.000 254 Control64.0 0.13 Ample nitrogen WRKY17 I 100.9 — 0.23 0.033 192 WRKY17 2 114.3| 0.026 147 0.22 0.054 183 WRKY17 3 114.8 0.023 148 0.23 0.040 192Control 77.6 0.12

ZAT11:

Three independent ZAT11 overexpressing lines showed a significantincrease in NupE and UI under both limiting and ample N conditions.

TABLE 13 Enhanced nitrogen uptake in plants ectopically expressing ZAT11Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incr Limitingnitrogen ZAT11 1 128.1 0.000 200 0.34 0.000 262 ZAT11 2 130.1 0.000 2030.32 0.000 246 ZAT11 3 117.1 0.000 183 0.38 0.000 292 Control 64.0 0.13Ample nitrogen ZAT11 1 115.4 0.019 149 0.25 0.007 208 ZAT11 2 125.60.000 162 0.27 0.001 225 ZAT11 3 146.6 0.000 189 0.28 0.000 233 Control77.6 0.12

HMGB14:

Three independent HMGB14 lines showed an increase in UI under limiting Nconditions; one of these lines also showed an increase in UI under ampleN conditions and an increase in NupE under limiting N conditions.

TABLE 14 Enhanced nitrogen uptake in plants ectopically expressingHMGB14 Gene Event NupE UI Name # Ave. p val % incr Ave. p val % incrLimiting nitrogen HMGB14 1 100.4 0.22 .019 157 HMGB14 2 86.3 0.23 .016164 HMGB14 3 149.9 0.001 178 0.20 .088 143 Control 84.2 0.14 Amplenitrogen HMGB14 1 29.0 0.27 HMGB14 2 27.8 0.28 HMGB14 3 50.9 0.35 .010194 Control 39.7 0.18

Example VI. Plant Transformation Methods

Crop species that overexpress polypeptides of the instant descriptionmay produce plants with increased photosynthetic resource use efficiencyand/or yield. Thus, polynucleotide sequences listed in the SequenceListing recombined into, for example, one of the expression vectors ofthe instant description, or another suitable expression vector, may betransformed into a plant for the purpose of modifying plant traits forthe purpose of improving yield, quality, and/or photosynthetic resourceuse efficiency. The expression vector may contain a constitutive,tissue-enhanced or inducible promoter operably linked to thepolynucleotide. The cloning vector may be introduced into a variety ofplants by means well known in the art such as, for example, direct DNAtransfer or Agrobacterium tumefaciens-mediated transformation.

Transformation of Monocots.

Cereal plants including corn, wheat, rice, sorghum, barley, or othermonocots may be transformed with the present polynucleotide sequences,including monocot or eudicot-derived sequences such as those presentedin the present Tables, cloned into a vector such as pGA643 andcontaining a kanamycin-resistance marker, and expressed constitutivelyunder, for example, the CaMV35S or COR15 promoters, or withtissue-enhanced or inducible promoters. The expression vectors may beone found in the Sequence Listing, or any other suitable expressionvector may be similarly used. For example, pMEN020 may be modified toreplace the NptII coding region with the BAR gene of Streptomyceshygroscopicus that confers resistance to phosphinothricin. The KpnI andBglII sites of the Bar gene are removed by site-directed mutagenesiswith silent codon changes.

The cloning vector may be introduced into a variety of cereal plants bymeans well known in the art including direct DNA transfer orAgrobacterium tumefaciens-mediated transformation. The latter approachmay be accomplished by a variety of means, including, for example, thatof U.S. Pat. No. 5,591,616, in which monocotyledon callus is transformedby contacting dedifferentiating tissue with the Agrobacterium containingthe cloning vector.

The sample tissues are immersed in a suspension of 3×10⁻⁹ cells ofAgrobacterium containing the cloning vector for 3-10 minutes. The callusmaterial is cultured on solid medium at 25° C. in the dark for severaldays. The calli grown on this medium are transferred to a RegenerationMedium. Transfers are continued every two to three weeks (two or threetimes) until shoots develop. Shoots are then transferred toShoot-Elongation Medium every 2-3 weeks. Healthy looking shoots aretransferred to Rooting Medium and after roots have developed, the plantsare placed into moist potting soil.

The transformed plants are then analyzed for the presence of the NPTIIgene/kanamycin resistance by ELISA, using the ELISA NPTII kit from5Prime-3Prime Inc. (Boulder, Colo.).

It is also routine to use other methods to produce transgenic plants ofmost cereal crops (Vasil, 1994. Plant Mol. Biol. 25: 925-937) such ascorn, wheat, rice, sorghum (Cassas et al., 1993. Proc. Natl. Acad. Sci.USA 90: 11212-11216), and barley (Wan and Lemeaux, 1994. Plant Physiol.104: 37-48). DNA transfer methods such as the microprojectile method canbe used for corn (Fromm et al., 1990. Bio/Technol. 8: 833-839;Gordon-Kamm et al., 1990. Plant Cell 2: 603-618; Ishida, 1990. NatureBiotechnol. 14:745-750), wheat (Vasil et al., 1992. Bio/Technol.10:667-674; Vasil et al., 1993. Bio/Technol. 11:1553-1558; Weeks et al.,1993. Plant Physiol. 102:1077-1084), and rice (Christou, 1991.Bio/Technol. 9:957-962; Hiei et al., 1994. Plant J. 6:271-282; Aldemitaand Hodges, 1996. Planta 199: 612-617; and Hiei et al., 1997. Plant Mol.Biol. 35:205-218). For most cereal plants, embryogenic cells derivedfrom immature scutellum tissues are the preferred cellular targets fortransformation (Hiei et al., 1997. supra; Vasil, 1994. supra). Fortransforming corn embryogenic cells derived from immature scutellartissue using microprojectile bombardment, the A188XB73 genotype is thepreferred genotype (Fromm et al., 1990. Bio/Technol. 8: 833-839;Gordon-Kamm et al., 1990. supra). After microprojectile bombardment thetissues are selected on phosphinothricin to identify the transgenicembryogenic cells (Gordon-Kamm et al., 1990. supra). Transgenic plantsfrom transformed host plant cells may be regenerated by standard cornregeneration techniques (Fromm et al., 1990. Bio/Technol. 8: 833-839;Gordon-Kamm et al., 1990. supra).

Transformation of Dicots.

It is now routine to produce transgenic plants using most eudicot plants(see U.S. Pat. No. 8,273,954 (Rogers et al.) issued Sep. 25, 2012;Weissbach and Weissbach, 1989. Methods for Plant Molecular Biology,Academic Press; Gelvin et al., 1990. Plant Molecular Biology Manual,Kluwer Academic Publishers; Herrera-Estrella et al., 1983. Nature 303:209; Bevan, 1984. Nucleic Acids Res. 12: 8711-8721; and Klee, 1985.Bio/Technology 3: 637-642). Methods for analysis of traits are routinein the art and examples are disclosed above.

Numerous protocols for the transformation of tomato and soy plants havebeen previously described, and are well known in the art. Gruber et al.,in Glick and Thompson, 1993. Methods in Plant Molecular Biology andBiotechnology. eds., CRC Press, Inc., Boca Raton, describe severalexpression vectors and culture methods that may be used for cell ortissue transformation and subsequent regeneration. For soybeantransformation, methods are described by Mild et al., 1993. in Methodsin Plant Molecular Biology and Biotechnology, p. 67-88, Glick andThompson, eds., CRC Press, Inc., Boca Raton; and U.S. Pat. No.5,563,055, (Townsend and Thomas), issued Oct. 8, 1996.

There are a substantial number of alternatives to Agrobacterium-mediatedtransformation protocols, other methods for the purpose of transferringexogenous genes into soybeans or tomatoes. One such method ismicroprojectile-mediated transformation, in which DNA on the surface ofmicroprojectile particles is driven into plant tissues with a biolisticdevice (see, for example, Sanford et al., 1987. Part. Sci. Technol.5:27-37; Sanford, 1993. Methods Enzymol. 217: 483-509; Christou et al.,1992. Plant. J. 2: 275-281; Klein et al., 1987. Nature 327: 70-73; U.S.Pat. No. 5,015,580 (Christou et al), issued May 14, 1991; and U.S. Pat.No. 5,322,783 (Tomes et al.), issued Jun. 21, 1994).

Alternatively, sonication methods (see, for example, Zhang et al., 1991.Bio/Technology 9: 996-997); direct uptake of DNA into protoplasts usingCaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine (see, forexample, Hain et al., 1985. Mol. Gen. Genet. 199: 161-168; Draper etal., 1982. Plant Cell Physiol. 23: 451-458); liposome or spheroplastfusion (see, for example, Deshayes et al., 1985. EMBO J., 4: 2731-2737;Christou et al., 1987. Proc. Natl. Acad. Sci. USA 84: 3962-3966); andelectroporation of protoplasts and whole cells and tissues (see, forexample, Donn et al. 1990. in Abstracts of VIIth International Congresson Plant Cell and Tissue Culture IAPTC, A2-38: 53; D'Halluin et al.,1992. Plant Cell 4: 1495-1505; and Spencer et al., 1994. Plant Mol.Biol. 24: 51-61) have been used to introduce foreign DNA and expressionvectors into plants.

After a plant or plant cell is transformed (and the transformed hostplant cell then regenerated into a plant), the transformed plant maypropagated vegetatively or it may be crossed with itself or a plant fromthe same line, a non-transformed or wild-type plant, or anothertransformed plant from a different transgenic line of plants. Crossingprovides the advantages of producing new and often stable transgenicvarieties. Genes and the traits they confer that have been introducedinto a tomato or soybean line may be moved into distinct line of plantsusing traditional backcrossing techniques well known in the art.Transformation of tomato plants may be conducted using the protocols ofKoornneef et al, 1986. In Tomato Biotechnology: Alan R. Liss, Inc.,169-178, and in U.S. Pat. No. 6,613,962, the latter method described inbrief here. Eight day old cotyledon explants are precultured for 24hours in Petri dishes containing a feeder layer of Petunia hybridasuspension cells plated on MS medium with 2% (w/v) sucrose and 0.8% agarsupplemented with 10 μM-naphthalene acetic acid and 4.4 μM6-benzylaminopurine. The explants are then infected with a dilutedovernight culture of Agrobacterium tumefaciens containing an expressionvector comprising a polynucleotide of the instant description for 5-10minutes, blotted dry on sterile filter paper and cocultured for 48 hourson the original feeder layer plates. Culture conditions are as describedabove. Overnight cultures of Agrobacterium tumefaciens are diluted inliquid MS medium with 2% (w/v/) sucrose, pH 5.7, to an OD₆₀₀ of 0.8.

Following cocultivation, the cotyledon explants are transferred to Petridishes with selective medium comprising MS medium with 4.56 μM zeatin,67.3 μM vancomycin, 418.9 μM cefotaxime and 171.6 μM kanamycin sulfate,and cultured under the culture conditions described above. The explantsare subcultured every three weeks onto fresh medium. Emerging shoots aredissected from the underlying callus and transferred to glass jars withselective medium without zeatin to form roots. The formation of roots ina kanamycin sulfate-containing medium is a positive indication of asuccessful transformation.

Transformation of soybean plants may be conducted using the methodsfound in, for example, U.S. Pat. No. 5,563,055 (Townsend et al., issuedOct. 8, 1996), described in brief here. In this method soybean seed issurface sterilized by exposure to chlorine gas evolved in a glass belljar. Seeds are germinated by plating on 1/10 strength agar solidifiedmedium without plant growth regulators and culturing at 28° C. with a 16hour day length. After three or four days, seed may be prepared forcocultivation. The seedcoat is removed and the elongating radicleremoved 3-4 mm below the cotyledons.

Eucalyptus is now considered an important crop that is grown for exampleto provide feedstocks for the pulp and paper and biofuel markets. Thisspecies is also amenable to transformation as described in PCT patentpublication WO/2005/032241.

Crambe has been recognized as a high potential oilseed crop that may begrown for the production of high value oils. An efficient method fortransformation of this species has been described in PCT patentpublication WO 2009/067398 A1.

Overnight cultures of Agrobacterium tumefaciens harboring the expressionvector comprising a polynucleotide of the instant description are grownto log phase, pooled, and concentrated by centrifugation. Inoculationsare conducted in batches such that each plate of seed was treated with anewly resuspended pellet of Agrobacterium. The pellets are resuspendedin 20 ml inoculation medium. The inoculum is poured into a Petri dishcontaining prepared seed and the cotyledonary nodes are macerated with asurgical blade. After 30 minutes the explants are transferred to platesof the same medium that has been solidified. Explants are embedded withthe adaxial side up and level with the surface of the medium andcultured at 22° C. for three days under white fluorescent light. Theseplants may then be regenerated according to methods well established inthe art, such as by moving the explants after three days to a liquidcounter-selection medium (see U.S. Pat. No. 5,563,055).

The explants may then be picked, embedded and cultured in solidifiedselection medium. After one month on selective media transformed tissuebecomes visible as green sectors of regenerating tissue against abackground of bleached, less healthy tissue. Explants with green sectorsare transferred to an elongation medium. Culture is continued on thismedium with transfers to fresh plates every two weeks. When shoots are0.5 cm in length they may be excised at the base and placed in a rootingmedium.

Experimental Methods; Transformation of Arabidopsis.

Transformation of Arabidopsis is performed by an Agrobacterium-mediatedprotocol based on the method of Bechtold and Pelletier, 1998. Unlessotherwise specified, all experimental work is performed using theColumbia ecotype.

Plant Preparation.

Arabidopsis seeds are gas sterilized and sown on plates with mediacontaining 80% MS with vitamins, 0.3% sucrose and 1% Bacto™ agar. Theplates are placed at 4° C. in the dark for the days then transferred to24 hour light at 22° C. for seven days. After 7 days the seedlings aretransplanted to soil, placing individual seedlings in each pot. Theprimary bolts are cut off a week before transformation to break apicaldominance and encourage auxiliary shoots to form. Transformation istypically performed at 4-5 weeks after sowing.

Bacterial Culture Preparation.

Agrobacterium stocks are inoculated from single colony plates or fromglycerol stocks and grown with the appropriate antibiotics untilsaturation. On the morning of transformation, the saturated cultures arecentrifuged and bacterial pellets are re-suspended in Infiltration Media(0.5× MS, 1× Gamborg's Vitamins, 5% sucrose, 200 μl/L Silwet® L77) untilan A₆₀₀ reading of 0.8 is reached.

Transformation and Harvest of Transgenic Seeds.

The Agrobacterium solution is poured into dipping containers. All flowerbuds and rosette leaves of the plants are immersed in this solution for30 seconds. The plants are laid on their side and wrapped to keep thehumidity high. The plants are kept this way overnight at 22° C. and thenthe pots are turned upright, unwrapped, and moved to the growth racks.In most cases, the transformation process is repeated one week later toincrease transformation efficiency.

The plants are maintained on the growth rack under 24-hour light untilseeds are ready to be harvested. Seeds are harvested when 80% of thesiliques of the transformed plants are ripe (approximately five weeksafter the initial transformation). This seed is deemed T₀ seed, since itis obtained from the T₀ generation, and is later plated on selectionplates (either kanamycin or sulfonamide). Resistant plants that areidentified on such selection plates comprise the T1 generation, fromwhich transgenic seed comprising an expression vector of interest may bederived.

Example VII. Identification of Crop Plants with Enhanced Nitrogen Uptake

Evaluating Transgenic Crop Plants for Enhanced Nitrogen Use Efficiencyand Nitrogen Uptake Efficiency (NupE) Under Field Conditions.

In field studies, the nitrogen use efficiency (NUE) in crop productionsystems can be computed using a number of methods (Table 15; Dobermann,2005, digitalcommons.unl.edu/agronomyfacpub/316). In field studies,these parameters are either calculated based on differences in cropyield and total N uptake with aboveground biomass between fertilizedplots and an unfertilized control (the “difference method”) or by using¹⁵N-labeled fertilizers to estimate crop and soil recovery of applied N.The time scale in these studies is usually one cropping season while thespatial scale for measurement is mostly a field or plot. Because eachparameter can differ in its value in interpreting the data, fieldresearch on fertilizer-N efficiency should include measurements ofseveral parameters to assess causes of variation in NUE. The “differencemethod” is simple and cost efficient procedure which makes itparticularly suitable for field research. The use of labeled ¹⁵N totrace the fate of applied N is a precise but expensive method that isonly applicable in scientific experiments and is not generally used infield studies.

TABLE 15 Agronomic indices of N use efficiency NUE index CalculationInterpretation NUE - Nitrogen use NUE = Y_(N)/F_(N) Most important forfarmers because it efficiency (also called integrates the use efficiencyof both partial factor indigenous and applied N resources: productivityof applied NUE = (Y₀/F_(N)) + AE_(N) N (PFP_(N)) (kg harvest Increasingindigenous soil N (Y₀) and product per kg N the efficiency of applied N(AE_(N)) are applied) equally important for improving NUE Limitedpotential for identifying specific constraints or promising managementstrategies AE_(N) = Agronomic AE_(N) = (Y_(N) − Y₀)/F_(N) AE_(N) is theproduct of the efficiency of efficiency of applied N recovery fromapplied N and the N (kg yield increase efficiency with which the plantuses per kg N applied) each additional unit of N acquired: AE_(N) =RE_(N) × PE_(N) AE_(N) can be increased by N, crop, and soil managementpractices that affect RE_(N), PE_(N), or both. RE_(N) = Crop recoveryRE_(N) = (U_(N) − U₀)/F_(N) RE_(N) depends on the congruence efficiencyof applied between plant N demand and the N (kg increase in N quantityof N released from applied N. uptake per kg N RE_(N) is affected by theN application applied) method (amount, timing, placement, N form) aswell as by factors that determine the size of the crop N sink (genotype,climate, plant density, abiotic/biotic stresses). PE_(N) = PhysiologicalPE_(N) = (Y_(N) − PE_(N) represents the ability of a plant to efficiencyof applied Y₀)/(U_(N) −U₀) transform N acquired from fertilizer N (kgyield increase into economic yield (grain). per kg increase in N PE_(N)depends on genotypic uptake from fertilizer) characteristics (e.g.,harvest index), environmental and management factors, particularlyduring reproductive growth. Low PE_(N) suggests sub-optimal growth(nutrient deficiencies, drought stress, heat stress, mineral toxicities,pests). FN-amount of (fertilizer) N applied (kg ha-1) YN-crop yield withapplied N (kg ha-1) Y0-crop yield (kg ha-1) in a control treatment withno N UN-total plant N uptake in aboveground biomass at maturity (kgha-1) in a plot that received N U0-the total N uptake in abovegroundbiomass at maturity (kg ha-1) in a plot that received no N

Example VIII. Embodiments of the Description

The contents and teachings of the instant application and each of theinformation sources provided herein can be relied on and used to makeand use embodiments of the instant disclosure. Although particularembodiments are described, equivalent embodiments may be used topractice the compositions and methods of the instant description,embodiments, and claims.

The instant disclosure pertains to, but is not limited by, the followingembodiments:

1. A method of altering architecture or physiology of a root tissue orgreen tissue in a plant, comprising:

-   -   (a) introducing into a plant cell a nucleic acid construct        comprising a constitutive, or a tissue-enhanced promoter that        preferentially regulates expression of a polypeptide in a root,        root cap, root meristem, root vasculature, vascular, and/or        green tissue, wherein:    -   said polypeptide is at least 30%, at least 31%, at least 32%, at        least 33%, at least 34%, at least 35%, at least 36%, at least        37%, at least 38%, at least 39%, at least 40%, at least 41%, at        least 42%, at least 43%, at least 44%, at least 45%, at least        46%, at least 47%, at least 48%, at least 49%, at least 50%, at        least 51%, at least 52%, at least 53%, at least 54%, at least        55%, at least 56%, at least 57%, at least 58%, at least 59%, at        least 60%, at least 61%, at least 62%, at least 63%, at least        64%, at least 65%, at least 66%, at least 67%, at least 68%, at        least 69%, at least 70%, at least 71%, at least 72%, at least        73%, at least 74%, at least 75%, at least 76%, at least 77%, at        least 78%, at least 79%, at least 90%, at least 81%, at least        82%, at least 83%, at least 84%, at least 85%, at least 86%, at        least 87%, at least 88%, at least 89%, at least 90%, at least        91%, at least 92%, at least 93%, at least 94%, at least 95% or        96%, at least 97%, at least 98%, or at least 99%, or about 100%        identical to SEQ ID NO:2n, where n=1 to 1131 or SEQ ID NO: 2263        to 2312;    -   wherein said regulation of expression of the polypeptide        increases nitrogen uptake, assimilation, nitrogen uptake        efficiency (NUpE), nitrogen usage index (UI), and/or seed yield        in the transgenic plant as compared to a control or reference        plant that does not comprise the a nucleic acid construct; and    -   b. optionally, identifying a transgenic plant that has greater        nitrogen uptake, assimilation, nitrogen uptake efficiency        (NUpE), nitrogen usage index (UI), and/or greater seed yield, as        compared to a control or reference plant.        2. A method for producing a plant that has enhanced nitrogen        uptake, assimilation, nitrogen uptake efficiency (NUpE),        nitrogen usage index (UI), and/or seed yield, the method        comprising:    -   (a) providing a plant with a nucleic acid construct comprising a        constitutive promoter, or a tissue-enhanced promoter that        preferentially regulates expression of a polypeptide in a root,        root cap, root meristem, root vasculature, vascular, and/or        green tissue, wherein:    -   said polypeptide is at least 30%, at least 31%, at least 32%, at        least 33%, at least 34%, at least 35%, at least 36%, at least        37%, at least 38%, at least 39%, at least 40%, at least 41%, at        least 42%, at least 43%, at least 44%, at least 45%, at least        46%, at least 47%, at least 48%, at least 49%, at least 50%, at        least 51%, at least 52%, at least 53%, at least 54%, at least        55%, at least 56%, at least 57%, at least 58%, at least 59%, at        least 60%, at least 61%, at least 62%, at least 63%, at least        64%, at least 65%, at least 66%, at least 67%, at least 68%, at        least 69%, at least 70%, at least 71%, at least 72%, at least        73%, at least 74%, at least 75%, at least 76%, at least 77%, at        least 78%, at least 79%, at least 90%, at least 81%, at least        82%, at least 83%, at least 84%, at least 85%, at least 86%, at        least 87%, at least 88%, at least 89%, at least 90%, at least        91%, at least 92%, at least 93%, at least 94%, at least 95% or        96%, at least 97%, at least 98%, or at least 99%, or about 100%        identical to SEQ ID NO:2n, where n=1 to 1131 or SEQ ID NO: 2263        to 2312;    -   wherein said regulation of expression of the polypeptide        increases nitrogen uptake in the transgenic plant as compared to        a control or reference plant that does not comprise the a        nucleic acid construct; and    -   b. optionally, identifying a transgenic plant that has greater        nitrogen uptake, assimilation, nitrogen uptake efficiency        (NUpE), nitrogen usage index (UI), and/or greater seed yield, as        compared to a control or reference plant.        3. The method of embodiment 2, wherein said providing the plant        with the nucleic acid construct is accomplished by transforming        a plant with the nucleic acid construct.        4. The method of embodiment 2, wherein said providing the plant        with the nucleic acid construct is accomplished by crossing the        plant with a plant that comprises the nucleic acid construct.        5. A method for producing a plant that has enhanced nitrogen        uptake, assimilation, nitrogen uptake efficiency (NUpE),        nitrogen usage index (UI), and/or greater seed yield in the        plant or a part of the plant comprising:    -   a. providing a constitutive promoter, or a root, root cap, root        meristem, root vasculature, vascular, and/or green        tissue-enhanced promoter that is capable of up-regulating gene        expression in the root, root cap, root meristem, root        vasculature, vascular, and/or green tissue structure, organ, or        plant part of the plant;    -   b. providing a suppressor of gene expression capable of        suppressing expression of an endogenous polynucleotide and its        encoded endogenous polypeptide;    -   wherein said polypeptide is at least 30%, at least 31%, at least        32%, at least 33%, at least 34%, at least 35%, at least 36%, at        least 37%, at least 38%, at least 39%, at least 40%, at least        41%, at least 42%, at least 43%, at least 44%, at least 45%, at        least 46%, at least 47%, at least 48%, at least 49%, at least        50%, at least 51%, at least 52%, at least 53%, at least 54%, at        least 55%, at least 56%, at least 57%, at least 58%, at least        59%, at least 60%, at least 61%, at least 62%, at least 63%, at        least 64%, at least 65%, at least 66%, at least 67%, at least        68%, at least 69%, at least 70%, at least 71%, at least 72%, at        least 73%, at least 74%, at least 75%, at least 76%, at least        77%, at least 78%, at least 79%, at least 90%, at least 81%, at        least 82%, at least 83%, at least 84%, at least 85%, at least        86%, at least 87%, at least 88%, at least 89%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95% or 96%, at least 97%, at least 98%, or at least 99%, or        about 100% identical to SEQ ID NO:2n, where n=1 to 1131 or SEQ        ID NO: 2263 to 2312;    -   c. providing a target plant with at least one nucleic acid        construct to produce a transgenic plant, wherein the at least        one nucleic acid construct comprises the constitutive or        tissue-enhanced promoter and the suppressor of gene expression;    -   wherein the tissue-enhanced promoter increases expression of the        suppressor of gene expression in the structure, organ, or plant        part of the transgenic plant of the transgenic plant, which        results in decreased expression of the endogenous polynucleotide        and its encoded endogenous polypeptide; and said decreased        expression of the endogenous polypeptide increases nitrogen        uptake, assimilation, nitrogen uptake efficiency (NUpE),        nitrogen usage index (UI), and/or seed yield in the transgenic        plant as compared to a control or reference plant that does not        comprise the a nucleic acid construct; and    -   d. optionally, identifying a transgenic plant that has greater        nitrogen uptake, assimilation, nitrogen uptake efficiency        (NUpE), nitrogen usage index (UI), and/or greater seed yield, as        compared to the control or reference plant.        6. The method of embodiment 5, wherein the suppressor of gene        expression is an RNAi (RNA interference) molecule, a small        interfering RNA (siRNA) molecule, a small hairpin RNA (shRNA)        molecule, a microRNA (miRNA) molecule, an antisense molecule, a        cosuppression directing nucleic acid, a nucleic acid encoding a        ribozyme, a nucleic acid encoding a deoxyribozyme (DNAzyme), a        nucleic acid encoding a transcription factor suppressor, or a        triple helix oligonucleotide that decreases the expression of        the polynucleotide.        7. The method of embodiment 5, wherein the tissue-enhanced        promoter is selected from the group consisting of SEQ ID NOs:        2313 to 2372.        8. A method for producing a plant that has enhanced nitrogen        uptake in the plant or a part of the plant, comprising:    -   a. growing a plant in a medium containing:        -   a limiting concentration of nitrogen that limits growth of            the plant; or an ample concentration of nitrogen that does            not limit growth of the plant;    -   b. identifying a polypeptide the expression of which is higher        in a part of the plant including a root, root cap, root        meristem, root vasculature, vascular, leaf, and/or green tissue        structure or organ of a nitrogen-limited plant grown in the        limiting concentration of nitrogen than in a corresponding root,        root cap, root meristem, root vasculature, vascular, leaf,        and/or green tissue structure or organ of a nitrogen-ample plant        grown in the ample concentration of nitrogen;    -   d. identifying a polynucleotide that encodes the polypeptide;    -   c. identifying a constitutive promoter, or a root, root cap,        root meristem, root vasculature, vascular, and/or green        tissue-enhanced promoter that is capable of up-regulating        protein expression in a root, root cap, root meristem, root        vasculature, vascular, leaf, and/or green tissue structure or        organ;    -   e. introducing into a target plant at least one recombinant        nucleic acid construct to produce a transgenic plant, and the at        least one recombinant nucleic acid construct comprises the        polynucleotide and the constitutive or tissue-enhanced promoter;        -   wherein the tissue-enhanced promoter regulates transcription            of the polynucleotide, and said transcriptional regulation            is preferentially enhanced in the root, root cap, root            meristem, root vasculature, vascular, and/or green tissue            structure or organ of the transgenic plant;        -   wherein the preferentially enhanced expression of the            polypeptide in the transgenic plant or in the root, root            cap, root meristem, root vasculature, vascular, and/or green            tissue structure or organ of the transgenic plant increases            nitrogen uptake, assimilation, nitrogen uptake efficiency            (NUpE), nitrogen usage index (UI), and/or seed yield in the            transgenic plant as compared to a control or reference plant            that does not comprise the a nucleic acid construct; and    -   f. optionally, selecting a transgenic plant that has greater        nitrogen uptake, assimilation, nitrogen uptake efficiency        (NUpE), nitrogen usage index (UI), and/or greater seed yield        than the control plant.        9. A method for producing a plant that has enhanced nitrogen        uptake in the plant or a part of the plant, comprising:    -   a. growing a plant in a medium containing:        -   a limiting concentration of nitrogen that limits growth of            the plant; or an ample concentration of nitrogen that does            not limit growth of the plant;    -   b. identifying a polypeptide the expression of which is lower in        a part of the plant including a root, root cap, root meristem,        root vasculature, vascular, leaf, and/or green tissue structure        or organ of a nitrogen-limited plant grown in the limiting        concentration of nitrogen than in a corresponding root, root        cap, root meristem, root vasculature, vascular, leaf, and/or        green tissue structure or organ of a nitrogen-ample plant grown        in the ample concentration of nitrogen;    -   d. identifying a polynucleotide that encodes the polypeptide;    -   c. identifying a constitutive promoter, or a root, root cap,        root meristem, root vasculature, vascular, and/or green        tissue-enhanced promoter that is capable of up-regulating        protein expression in a root, root cap, root meristem, root        vasculature, vascular, leaf, and/or green tissue structure or        organ;    -   e. introducing into a target plant at least one recombinant        nucleic acid construct to produce a transgenic plant, and the at        least one recombinant nucleic acid construct comprises the        polynucleotide and the constitutive or tissue-enhanced promoter;        -   wherein the tissue-enhanced promoter regulates transcription            of the polynucleotide, and said transcriptional regulation            is preferentially enhanced in the root, root cap, root            meristem, root vasculature, vascular, and/or green tissue            structure or organ of the transgenic plant;        -   wherein the preferentially enhanced expression of the            polypeptide in the transgenic plant or in the root, root            cap, root meristem, root vasculature, vascular, and/or green            tissue structure or organ of the transgenic plant increases            nitrogen uptake, assimilation, nitrogen uptake efficiency            (NUpE), nitrogen usage index (UI), and/or seed yield in the            transgenic plant as compared to a control or reference plant            that does not comprise the a nucleic acid construct; and    -   f. optionally, selecting a transgenic plant that has greater        nitrogen uptake, assimilation, nitrogen uptake efficiency        (NUpE), nitrogen usage index (UI), and/or greater seed yield        than the control plant.        10. The method of embodiment 8 or embodiment 9, wherein the        limiting concentration of nitrogen in the medium is a total        nitrogen content of 2 mM nitrogen and the ample concentration of        nitrogen is a total nitrogen content of 10 mM nitrogen.        11. The method of embodiment 8, embodiment 9, or embodiment 10,        wherein the polypeptide is at least 30%, at least 31%, at least        32%, at least 33%, at least 34%, at least 35%, at least 36%, at        least 37%, at least 38%, at least 39%, at least 40%, at least        41%, at least 42%, at least 43%, at least 44%, at least 45%, at        least 46%, at least 47%, at least 48%, at least 49%, at least        50%, at least 51%, at least 52%, at least 53%, at least 54%, at        least 55%, at least 56%, at least 57%, at least 58%, at least        59%, at least 60%, at least 61%, at least 62%, at least 63%, at        least 64%, at least 65%, at least 66%, at least 67%, at least        68%, at least 69%, at least 70%, at least 71%, at least 72%, at        least 73%, at least 74%, at least 75%, at least 76%, at least        77%, at least 78%, at least 79%, at least 90%, at least 81%, at        least 82%, at least 83%, at least 84%, at least 85%, at least        86%, at least 87%, at least 88%, at least 89%, at least 90%, at        least 91%, at least 92%, at least 93%, at least 94%, at least        95% or 96%, at least 97%, at least 98%, or at least 99%, or        about 100% identical to SEQ ID NO:2n, where n=1 to 1131        12. A method for enhancing nitrogen uptake in a crop plant, the        method comprising:    -   providing a transgenic crop plant that comprises at least one        nucleic acid construct, wherein the nucleic acid construct        comprises a promoter and a polynucleotide; and    -   the promoter is a constitutive promoter or a root, root cap,        root meristem, root vasculature, vascular, or green        tissue-enhanced promoter;    -   wherein the polynucleotide encodes a polypeptide is at least        30%, at least 31%, at least 32%, at least 33%, at least 34%, at        least 35%, at least 36%, at least 37%, at least 38%, at least        39%, at least 40%, at least 41%, at least 42%, at least 43%, at        least 44%, at least 45%, at least 46%, at least 47%, at least        48%, at least 49%, at least 50%, at least 51%, at least 52%, at        least 53%, at least 54%, at least 55%, at least 56%, at least        57%, at least 58%, at least 59%, at least 60%, at least 61%, at        least 62%, at least 63%, at least 64%, at least 65%, at least        66%, at least 67%, at least 68%, at least 69%, at least 70%, at        least 71%, at least 72%, at least 73%, at least 74%, at least        75%, at least 76%, at least 77%, at least 78%, at least 79%, at        least 90%, at least 81%, at least 82%, at least 83%, at least        84%, at least 85%, at least 86%, at least 87%, at least 88%, at        least 89%, at least 90%, at least 91%, at least 92%, at least        93%, at least 94%, at least 95% or 96%, at least 97%, at least        98%, or at least 99%, or about 100% identical to SEQ ID NO:2n,        where n=1 to 1131; and    -   wherein the promoter enhances expression of the polynucleotide        in the transgenic crop plant, or preferentially enhances        expression of the polynucleotide in a root, root cap, root        meristem, root vasculature, vascular, and/or green tissue        structure or organ of the transgenic crop plant, and said        preferential enhancement of expression increases nitrogen uptake        in the transgenic crop plant relative to a control or reference        plant that does not comprise the a nucleic acid construct.        13. A method for enhancing nitrogen uptake, assimilation,        nitrogen uptake efficiency (NUpE), nitrogen usage index (UI),        and/or seed yield in a crop plant, the method comprising:    -   providing a transgenic crop plant that comprises at least one        recombinant nucleic acid construct, wherein the nucleic acid        construct comprises a constitutive promoter or a root, root cap,        root meristem, root vasculature, vascular, and/or green        tissue-enhanced promoter, and a suppressor of gene expression        capable of suppressing expression of an endogenous        polynucleotide;    -   wherein the suppressor of gene expression inhibits expression of        the polynucleotide and its encoded endogenous polypeptide, and        the endogenous polypeptide is at least 30%, at least 31%, at        least 32%, at least 33%, at least 34%, at least 35%, at least        36%, at least 37%, at least 38%, at least 39%, at least 40%, at        least 41%, at least 42%, at least 43%, at least 44%, at least        45%, at least 46%, at least 47%, at least 48%, at least 49%, at        least 50%, at least 51%, at least 52%, at least 53%, at least        54%, at least 55%, at least 56%, at least 57%, at least 58%, at        least 59%, at least 60%, at least 61%, at least 62%, at least        63%, at least 64%, at least 65%, at least 66%, at least 67%, at        least 68%, at least 69%, at least 70%, at least 71%, at least        72%, at least 73%, at least 74%, at least 75%, at least 76%, at        least 77%, at least 78%, at least 79%, at least 90%, at least        81%, at least 82%, at least 83%, at least 84%, at least 85%, at        least 86%, at least 87%, at least 88%, at least 89%, at least        90%, at least 91%, at least 92%, at least 93%, at least 94%, at        least 95%, at least 96%, at least 97%, at least 98%, at least        99%, or about 100% identical to SEQ ID NO:2n, where n=1 to 1131        or SEQ ID NO: 2263-2312; and    -   wherein the constitutive or tissue-enhanced promoter increases        expression of the suppressor of gene expression in the        transgenic plant or the part of the transgenic plant, which        results in decreased expression of the endogenous polynucleotide        and its encoded endogenous polypeptide; and    -   said decreased expression of the endogenous polypeptide        increases nitrogen uptake, assimilation, nitrogen uptake        efficiency (NUpE), nitrogen usage index (UI), and/or seed yield        in the transgenic plant relative to the control plant.        14. The method of embodiment 13, wherein the suppressor of gene        expression is an RNAi (RNA interference) molecule, a small        interfering RNA (siRNA) molecule, a small hairpin RNA (shRNA)        molecule, a microRNA (miRNA) molecule, an antisense molecule, a        cosuppression directing nucleic acid, a nucleic acid encoding a        ribozyme, a nucleic acid encoding a deoxyribozyme (DNAzyme), a        nucleic acid encoding a transcription factor suppressor, or a        triple helix oligonucleotide that decreases the expression of        the polynucleotide.        15. The method of any of embodiments 13 or 14, wherein the plant        has higher nitrogen uptake, assimilation, nitrogen uptake        efficiency (NUpE), nitrogen usage index (UI), and/or seed yield.        16. The method of any of embodiments 13 to 15, wherein the        tissue-enhanced promoter is selected from the group consisting        of SEQ ID NOs: 2313 to 2349 or a promoter listed in Table 3.        17. A recombinant nucleic acid construct comprising a root, root        cap, root meristem, root vasculature, vascular, and/or green        tissue-enhanced promoter selected from the group consisting of        SEQ ID NOs: 2313 to 2372;    -   wherein the promoter regulates expression of a polynucleotide        that inhibits expression of an endogenous polypeptide that is at        least 30%, at least 31%, at least 32%, at least 33%, at least        34%, at least 35%, at least 36%, at least 37%, at least 38%, at        least 39%, at least 40%, at least 41%, at least 42%, at least        43%, at least 44%, at least 45%, at least 46%, at least 47%, at        least 48%, at least 49%, at least 50%, at least 51%, at least        52%, at least 53%, at least 54%, at least 55%, at least 56%, at        least 57%, at least 58%, at least 59%, at least 60%, at least        61%, at least 62%, at least 63%, at least 64%, at least 65%, at        least 66%, at least 67%, at least 68%, at least 69%, at least        70%, at least 71%, at least 72%, at least 73%, at least 74%, at        least 75%, at least 76%, at least 77%, at least 78%, at least        79%, at least 90%, at least 81%, at least 82%, at least 83%, at        least 84%, at least 85%, at least 86%, at least 87%, at least        88%, at least 89%, at least 90%, at least 91%, at least 92%, at        least 93%, at least 94%, at least 95% or 96%, at least 97%, at        least 98%, or at least 99%, or about 100% identical to SEQ ID        NO:2n, where n=1 to 1131, or to SEQ ID NO 2263 to 2312.        18. A transgenic crop plant produced by the method of any of        embodiments 1 to 16 or comprising a recombinant nucleic acid        construct of embodiment 17, wherein the transgenic plant has        enhanced nitrogen uptake, assimilation, nitrogen uptake        efficiency (NUpE), nitrogen usage index (UI), and/or seed yield,        as compared to the control or reference plant.        19. The transgenic crop plant of embodiment 18, wherein the        transgenic plant is selected from the group consisting of: a        non-leguminous plant, a monocot plant, a cereal plant, a maize        (corn) plant, a rice plant, a wheat plant, a barley plant, a        sorghum plant, a millet plant, an oat plant, a triticale plant,        a rye plant, a buckwheat plant, a fonio plant, and a quinoa        plant.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The present invention is not limited by the specific embodimentsdescribed herein. The invention now being fully described, it will beapparent to one of ordinary skill in the art that many changes andmodifications can be made thereto without departing from the spirit orscope of the appended claims. Modifications that become apparent fromthe foregoing description fall within the scope of the claims.

What is claimed is:
 1. A method of altering architecture or physiologyof a green tissue in a plant, comprising: (a) introducing into a plantcell a nucleic acid construct comprising a tissue-enhanced promoterconsisting of the nucleic acid sequence set forth in SEQ ID NO: 2324,that preferentially regulates expression of a polypeptide having theamino acid sequence set forth in SEQ ID NO: 76 in a green tissue,wherein regulation of expression of the polypeptide increases nitrogenuptake, assimilation, nitrogen uptake efficiency (NUpE), nitrogen usageindex (UI), and/or seed yield in a transgenic plant comprising the plantcell as compared to a control or reference plant that does not comprisethe nucleic acid construct; and (b) optionally, identifying thetransgenic plant comprising the plant cell.
 2. A method for producing aplant that has enhanced nitrogen uptake, assimilation, nitrogen uptakeefficiency (NUpE), nitrogen usage index (UI), and/or seed yield, themethod comprising: (a) providing a plant with a nucleic acid constructcomprising a tissue-enhanced promote consisting of the nucleic acidsequence set forth in SEQ ID NO: 2324, that preferentially regulatesexpression of a polypeptide having the amino acid sequence set forth inSEQ ID NO: 76 in a green tissue, wherein regulation of expression of thepolypeptide increases nitrogen uptake in a transgenic plant comprisingthe plant cell as compared to a control or reference plant that does notcomprise the nucleic acid construct; and (b) optionally, identifying thetransgenic plant comprising the plant cell.
 3. The method of claim 2,wherein the providing a plant with a nucleic acid construct comprisestransforming the plant with the nucleic acid construct.
 4. The method ofclaim 2, wherein the providing a plant with a nucleic acid constructcomprises crossing the plant with a second plant that comprises thenucleic acid construct.